Unit 2 Practical Microbiology and Infectious Diseases

Pearson
BTEC Level 3 National

Extended Certificate in
Applied Human
Biology
Unit 2
Practical Microbiology
and Infectious Diseases
Practical microbiology and infectious diseases 1
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Table of Contents
How to use this document 3
Features of this document 3
How you will be assessed 5
A Understand the classification and nature of microorganisms 7
A1 Characteristics of different microorganisms 8
A2 Methods of pathogenicity 15
A3 Classification strategies 24
B Examine the transmission and treatments of infectious disease 29
B1 Classification overview of infectious disease 29
B2 Transmission of infectious disease 34
B3 Infectious diseases 40
B4 Prevention and treatment of infectious diseases 50
C Explore the application of techniques to culture and identify microorganisms 56
C1 Health and safety 56
C2 Microscopy and staining techniques 63
C3 Culture of microorganisms 68
C4 Quantitative analysis of microbes 73
D: Investigate the effect of antimicrobial agents on the growth of microorganisms 78
D1 Investigating the substances that inhibit the growth of microorganisms 78
D2 Interpretation, analysis and evaluation of results 82
Think Future Skills 89
Think about it 90

How to use this document
Welcome to your Applied Human Biology course.
A BTEC National in Applied Human Biology will give you the opportunity to develop a range of skills that
will prepare you for the world of work, or for continued study at a higher level.
The number of units in your BTEC National qualification varies depending on the size of qualification
you are doing.
This document supports the specification and associated assessment guidance, it does not replace
them and should not be used in their place. Teachers should use their expertise and judgement re-
garding the teaching and delivery of this course and ensure that learners are taught all areas of con-
tent in sufficient depth in preparation for the external assessment. Every effort has been made to
cover as much of the specification as possible. This document does not indicate topics, question types
or activities that may come up in the external assessment and no member of the examination team
has been involved in its creation.
Features of this document

There are a number of different features throughout the document, designed to help you learn about
the topics in your course in different ways and understand it from multiple perspectives. Together
these features:
• explain what your learning is about

• help to build your knowledge
• help you to reflect on and evaluate your learning
• make you think beyond what you are reading about
• help you make connections between your learning and real-world workplace environments.
In addition, each feature has a specific purpose designed to support your learning.
Features that explain what your learning is about
Getting to know your unit

This section introduces the unit and explains how you will be assessed. It gives an overview of what will
be covered throughout the unit.
Features that help you build your knowledge
Worked example
The worked examples show the process you need to follow to solve a problem, such as a maths
or science equation. This will also help you to develop your understanding and your numeracy
and literacy skills.
Key points
Concise and simple definitions are provided for key words, phrases and concepts, allowing you to
have, at a glance, a clear understanding of the key ideas in the unit.

Features connected to your assessment
Assessment practice
These features give you the opportunity to practise some of the skills you will need when you
are assessed on the unit. They do not fully reflect the actual assessment tasks but will help you
prepare for them.
Features to help you reflect on and evaluate your learning
Pause point
Pause points give you the opportunity to review and reflect on your own learning. The ability to
reflect on your learning is a key skill you will need to develop and use throughout your life.
Hint and Expand
These points also give you suggestions to help cement your knowledge and indicate other areas
you can look at to expand it.
Case studies are used in the unit to allow you to apply the learning and knowledge from the unit to a
scenario from the workplace or industry. Case studies include questions to help you consider the wider
context of a topic.
Think future skills

This section includes a case study of someone working in the industry. They talk about their job role
and the skills they need. This comes with a Focusing your skills section, which gives suggestions for
how you can begin to develop the employability skills and experiences needed to be successful in a
career in your chosen sector. This is an excellent opportunity to build up your employability skills.

Getting to know your unit
Assessment When studying human biology, it is important that you have a good under-
standing of how microorganisms can cause infectious diseases in humans.
You will be assessed by a
You will study how infectious diseases can be transmitted from person to per-
series of assignments set by
son and explore the different treatments that can be given. You will also learn
your teacher/tutor.
how to culture and identify microorganisms safely in the laboratory and carry
out investigations into how different substances affect their growth.
How you will be assessed

In this unit, you will be assessed by a series of internally assessed tasks set by the unit teacher.
Throughout the unit, you will find assessment activities that may help you work towards your assess-
ment. Completing these activities will not mean you have achieved a particular grade, but the research
you carry out for them will be relevant and useful when you come to carry out your final assessment.
It is important to check that you have met the Pass grading criteria, shown in the table below, as you
work your way through the assignments.
To achieve a Merit or Distinction, you need to present your work in such a way that you met the criteria
for those grades.
The assignments set by your tutor will consist of several tasks designed to meet the criteria in the table
below.
Assessment criteria
This table shows what you must do in order to achieve a Pass, Merit or Distinction grade, and where you
can find activities to help you.
Pass Merit Distinction
Learning aim A: Understand the classification and nature of microorganisms
A.P1 A.M1 AB.D1
Explain the methods used to classify Analyse the virulence mecha- Evaluate the treatments of
microorganisms. nisms of microorganisms that the types of infectious dis-
cause infectious diseases and the ease and the current issues
A.P2 methods used to classify micro- in the development of these
Explain the role of the structures found organisms. treatments.
in microorganisms and the factors affect-
ing their growth and virulence that cause
infectious disease.
Learning aim B: Examine the transmission and treatments of infectious

disease
B.P3 B.M2
Describe the development of different Discuss the development of in-
types of disease. fectious diseases and their asso-
ciated prevention and treatment
B.P4 strategies.
Explain the prevention and treatment
strategies for the different types of infec-
tious disease,

Learning aim C: Explore the application of techniques to culture and identify microorganisms
C.P5 C.M3 C.D2
Carry out morphological studies, Compare the techniques used Correctly use aseptic enumer-
microscopy and staining tech- to identify and cultivate microor- ation techniques and make
niques to identify microorganisms. ganisms in terms of the quality of judgements on the accuracy of
results obtained. the procedure used.
C.P6 C.M4
Correctly select and use aseptic Correctly select and use aseptic
technique to cultivate microorgan- technique to grow and measure
isms. the growth of microorganisms, in-
cluding the use of serial dilutions.
Learning aim D: Investigate the effect of antimicrobial agents on the growth of microorganisms
D.P7 D.M5 D.D3
Plan and carry out an investigation Plan and carry out an investigation Evaluate the methods, tech-
independently, selecting an appro- independently, selecting an appro- niques and data collected to
priate method, into the effects of priate method, into the effects of determine the effect of anti-
antimicrobials on the growth of antimicrobials on the growth of microbials on the growth of
organisms. organisms with little or no con- microorganisms and the wider
tamination of results. impact on the functioning of an
organism.
D.P8 D.M6
Interpret data collected in order Analyse the growth of microorgan-
to reach a conclusion, consider- isms using data collected and in
ing the impact on prevention and relation to the factors investigated
treatment of disease. in order to reach valid conclusion,
making links to the impact on
the prevention and treatment of
disease.

Getting started
Microorganisms can cause disease in humans. Diseases causing microorganisms are called pathogens
and include some bacteria, fungi, protists, viruses and prions. Write down a list of different human
diseases caused by each of these pathogens. When you have completed this unit, add more diseases
to your list. Write down the different ways in which diseases can be transmitted from person to person.
When you have completed this unit, see if you can add more to your list.
A Understand the classification and

nature of microorganisms
Infectious diseases such as tuberculosis (TB), HIV and Ebola are caused by microorganisms. Microor-
ganisms are organisms that cannot be seen with the naked eye; you need to use a microscope to be
able to see them. Microorganisms include bacteria, viruses, prions, protists and fungi. Not all microor-
ganisms cause disease but those that do are referred to as pathogens.
All cells can be classified into one of six kingdoms of life. The six kingdoms are:
• Animals – these cells are eukaryotic with no cell wall

• Plants – these cells are eukaryotic with a cell wall made of cellulose
• Fungi – these cells are eukaryotic with a cell wall made of chitin
• Protists -these cells are single-celled eukaryotes that do not fit into one of the above kingdoms
• Eubacteria – these cells are prokaryotic with cell walls made of peptidoglycan
• Archaea – these cells are prokaryotic and can live in extreme conditions.
In this section, you will learn about the differences between eukaryotic cells and prokaryotic cells
and the characteristic features of the kingdoms containing disease-causing pathogens.
Key points
Microorganism – microscopic organisms including bacteria, fungi, viruses, prions and protists.
Pathogen – a microorganism that causes disease.
Eukaryotic cell – a cell that contains a nucleus and other membrane bound organelles such as
mitochondria.
Prokaryotic cell – a cell that does not contain a nucleus or other membrane bound organelles.

A1 Characteristics of different microorganisms
Prokaryotes
Physical characteristics
A prokaryotic cell is a cell with certain characteristic features, this group of cells includes bacteria. Bac-
terial cells have:
• no nucleus, their DNA is found as a single loop in the cytoplasm in a region called the nucleoid
• no membrane-bound organelles
• a cell wall made of peptidoglycan
• a cell surface membrane
• ribosomes that are smaller than those found in eukaryotic cells
• small additional rings of DNA called plasmids.
They sometimes have:
• a protective capsule (slime) layer around the cell wall which prevents the cell from drying out
• flagellum that allows the cell to move
• hair-like structures called pili that allow the cell to attach to surfaces or other cells.
Figure 2.1: Generalised structure of a bacterial cell
The cell wall

Bacterial cells are surrounded by a cell wall composed of peptidoglycan. Bacteria can be sorted into
two different groups (classified) based on the structure of their cell wall. Gram-positive bacteria have a
thick layer of peptidoglycan in their cell wall, whereas gram-negative bacteria cell walls have a thinner
layer of peptidoglycan. Most species of bacteria can be classified as Gram-positive or Gram-negative
using a practical technique called Gram staining. You will learn about this procedure later in this unit.

Growth characteristics
Reproduction
Bacteria reproduce using a process called binary fission. Bacteria are prokaryotes, which means that
their genome consists of a single loop of DNA, which lies in a region of the cytoplasm called the nu-
cleoid. Binary fission produces two new bacterial cells that are genetically identical to the parent cell.
Binary fission is asexual, this means that it does not involve the joining of gametes (sex cells).
The process of binary fission
1 The loop of DNA uncoils and is replicated to produce a second identical loop of DNA.
2 The cell increases in size and other structures are replicated including plasmid DNA and ribosomes.
3 The two loops of DNA are pulled to opposite sides of the cell.
4 The cell surface membrane begins to divide and a cross wall forms around each loop of DNA.
5 The cells separate to form two genetically identical daughter cells.
Figure 2.2: Bacterial reproduction by binary fission
Temperature
Bacteria can be classified by their optimum growth temperature. Each microorganism has a minimum,
optimum and maximum growth temperature.
Psychrophilic bacteria are capable of growth and reproduction at extremely low temperatures be-
tween -5oC and 20oC. The enzymes contained within psychrophiles have adapted to be optimally active
at low temperatures.
Mesophilic bacteria thrive in temperatures between 20oC and 45oC. Normal body temperature is ap-
proximately 37oC and, therefore, the bacteria that colonise the human body are mesophiles.
Thermophilic bacteria are heat-loving, they have an optimum growth temperature of between 50oC
and 70oC but can survive in very high temperatures. The enzymes within thermophilic bacteria are
thermostable, i.e. resistant to irreversible denaturing at high temperatures.

Growth curves
When bacteria are grown in a laboratory, the population produces a typical growth curve. In a closed
system, like a test tube, the population growth will follow a specific pattern as seen in Figure 2.3.
Growth curves have four characteristic phases.
1 Lag phase – the bacteria cells added to the growth medium increase in size and start to produce the
enzymes needed to use the nutrients present.
2 Exponential (log) phase – the bacteria are dividing by binary fission at a constant rate so that the
population size doubles with each generation time.
3 Stationary phase – the rate of binary fission decreases as population growth is limited by lack of
nutrients, build-up of waste or lack of space. In this phase, the reproduction rate is equal to the
death rate.
4 Death (decline phase) – the size of the bacterial population decreases because conditions are no
longer able to support reproduction of the cells.
Figure 2.3: Typical bacterial growth curve
Key points
Ribosome – organelle responsible for protein synthesis.

Genome – the genetic information (genes) found in a cell.
Growth medium – the substance used to grow a microorganism containing all the nutrients
required for growth. Can be solid, liquid or semi-solid.
Binary fission – a method of asexual reproduction used by prokaryotes.
Species – a group of organisms that share similar characteristics and can interbreed to produce
fertile offspring.

Eukaryotes
A eukaryotic cell has certain characteristic features. Animal and plant cells are eukaryotic. Eukaryotic
microorganisms include fungi and protists some of which can be human pathogens.
Eukaryotic cells have:
• a nucleus
• membrane-bound organelles
• a cell surface membrane
• ribosomes which are larger than those found in prokaryotic cells
• cell walls made of cellulose (in plant cells) and chitin (in fungi).
Protists
Protists are single-celled eukaryotic organisms; they have their DNA enclosed in a nucleus and have
membrane bound organelles. The features of protists are extremely broad with lots of variation be-
tween different species. Some species reproduce asexually, and some reproduce sexually. Some
species have cell walls while others have flexible cell membranes. Some protists obtain their nutrition
by being predators, some are parasites, and some synthesise their own nutrients. Most protists live in
a watery or moist environment and some species can live inside the human body and cause disease.
Protozoa are a type of protist that obtain their nutrition through the ingestion of organic matter, they
are referred to as heterotrophs. Other protists can carry out photosynthesis and so are not called pro-
tozoa. An example of a non-protozoan protist is algae. Malaria is a disease caused by a protist such as
Plasmodium falciparum, which can be transmitted by female Anopheles mosquitoes. Dysentery is also
caused by a protist (an amoeba) that lives in freshwater ponds.
Many protists are motile – they can move.
• Amoeba are a group of protists that move using specialised structures called pseudopodia. The
amoeba changes the structure of its cell to produce pseudopodia that then enable the cell to drag
itself along the environment.
• Some have one or more flagella, which they can rotate or whip to move, e.g. Euglena.
• Some protists such as Paramecium have a surface covered in rows of cilia that can beat in a
synchronised manner to coordinate movement through water.
• Some protists move towards (or away) from a stimulus. Algae, for example, show phototaxis, they
move towards sources of light.
Fungi
Fungi are eukaryotic organisms; the group includes yeasts, moulds and mushrooms. There are more
than 100 species of fungi that can cause disease in humans. As they are eukaryotic cells, fungi contain
a nucleus and membrane-bound organelles. Other features of fungi include:
• Can be single-celled such as yeast or multi-cellular such as mushrooms.

• Have cell walls that contain chitin.
• Obtain nutrition by feeding on and breaking down dead organic matter in the ecosystem.
• Most respire anaerobically but some fungi, such as yeast, can use both aerobic and anaerobic
respiration.
• Have hyphae which grow from the tips.
• Can be parasites (such as Tinea fungi which causes athlete’s foot).
• Use either asexual or sexual reproduction or by spores (asexual or sexual).

Asexual reproduction in yeast
Yeast cells are single-celled fungi that reproduce by a process called budding.
1 The parent cell forms a bud.

2 The bud grows as the DNA in the nucleus of the parent cell replicates.
3 The nucleus splits into two and one nucleus moves to the bud.
4 A wall forms between the bud and the parent cell.
5 The bud may remain attached to the parent cell or it may break away.
Figure 2.4: (a) Light micrograph of yeast cells Saccharomyces cerevisiae. Mag x340 (b) Colour enhanced SEM
of yeast cells Saccharomyces cerevisiae budding
Pause point
Distinguish between prokaryotic and eukaryotic cells.

Hint
Explain the similarities and differences between eukaryotic and prokaryotic cells referring to
specific examples.
Extend
Discuss how you could identify if a cell in an image is prokaryotic and eukaryotic.

Viruses and prions
Viruses and prions are not eukaryotic or prokaryotic. This is because viruses and prions are not living
and are not cells. They make up a third group of organisms known as the akaryotes. Akaryotes have no
cell structure, no cytoplasm or organelles.
Viruses
Viruses can infect all types of cell, there are viruses that can infect animals, plants, bacteria, protists
and fungi. Viruses:
• Consist of a capsid (protein coat) made of smaller units, called capsomeres.

• The capsid surrounds the nucleic acid, which can be DNA or RNA.
• Can have a lipid membrane called an envelope surrounding the capsid.
• Are small (smaller than bacteria) with diameters of 20-300nm.
• Can only reproduce inside a host cell.
• Can have a variety of shapes including helical, spherical and polyhedral.
Viruses can only reproduce inside a host cell. To reproduce, viruses attach to cells, insert their genetic
material into the host cell, take over the host cell machinery to replicate the nucleic acid and build new
viral particles. The viral particles then burst out of the host cell, destroying it. Each new virus particle
produced can infect other host cells. There are two types of viral reproduction cycle – lytic and
lysogenic.
Table 2.1: Lytic and lysogenic life cycles of viruses
Lytic Lysogenic
More common Rarer method of reproduction
Host cell is completely destroyed Host cell is not destroyed straight away, virus lies
dormant letting the cell multiply first
Viral nucleic acid replicates separately from host Viral nucleic acid replicates within the host DNA
cell DNA
One virus destroys one host cell One virus affects many cells as the virus is passed
along to new cells formed during cell division
Retroviruses
HIV (Human Immunodeficiency Virus) is a virus that can spread in bodily fluids such as blood and se-
men. It infects cells of the immune system called CD4 (T cells). Over time, the destruction of these cells
causes the development of AIDS (Acquired Immunodeficiency Syndrome). This weakens the immune
system and makes it difficult to fight off other infections. Opportunistic infections take advantage of
this and can lead to death.
HIV is an example of a type of virus called a retrovirus. HIV contains glycoprotein spikes on the enve-
lope, which attach to antigens on the surface of the T cells that it infects. Retroviruses such as HIV have
RNA as their genetic material and contain an enzyme called reverse transcriptase. When these viruses
replicate inside a host cell, the enzyme is used to produce complementary DNA (cDNA) from the RNA
nucleic acid. The DNA is incorporated into the host genome and may remain dormant there for sever-
al years. When activated, the virus directs the synthesis of new viral particles that use part of the host
cell’s membrane to form their envelopes. This destroys the host cell.

Figure 2.5: Replication of retroviruses such as HIV
Prions
Prions can also cause disease. Disease caused by prions is extremely rare and causes the nervous
system to lose function over time. Prion diseases have no known treatment and are always fatal. An
example of a neurodegenerative disease caused by a prion is variant Creutzfeldt-Jakob Disease (vCJD).
Prions are not living cells; they are proteins that are not folded correctly. Prions are stable and not
denatured by heat. The abnormal structure of proteins causes the protein to have infectious properties
and protects them from being broken down by normal cellular enzymes. When prions are transmitted
to a new organism, the prions cause proteins in that organism to misfold and aggregate (clump togeth-
er). These aggregates build up in the brain and nervous tissue. causing tissue damage. The damage
gets progressively worse over time.

Pause point
Discuss why viral and prionic diseases are more difficult to treat.
Hint
Link to the characteristic features of each group of microorganisms.
Extend
Discuss why prionic diseases are always fatal.
A2 Methods of pathogenicity
Key points
Pathogen – an infectious microorganism or agent that can cause disease.

Pathogenicity – the ability to cause disease.
Transmission – the spread of a pathogen.
Virulence –the degree of infection and damage caused by a pathogen.
Quantitative – measurements made where numerical data is collected.
Pathogenicity is used to describe the ability to cause disease. Whether transmission of the pathogen
to a host actually leads to the host developing disease depends on the virulence of the pathogen.
Virulence is the extent to which a pathogen is able to damage host cells and cause disease. A pathogen
can be classified as pathogenic but depending on conditions, may produce different levels of virulence.
Virulence can be measured on a scale from avirulent (not harmful) to highly virulent.
Avirulent Increasing virulence Highly

virulent
May cause infection Causes severe illness but May cause severe illness,
Not but will not cause most people can recover. multi-organ failure and
harmful serious long-lasting Risk to those with compro- even death in normally
illness mised immune system healthy individuals
Figure 2.6: Scale of virulence related to the properties of the diseases caused
Quantitative measures are typically used to measure virulence. The two most common measures of
virulence are the median infectious dose and median lethal dose. These measurements are carried out
in laboratories, often using animal models or simulations. The median infectious dose is the number of
pathogens required to cause infection in 50% of organisms infected with the pathogen. The median le-
thal dose is the number of pathogens required to cause death in 50% of infected organisms. The lower
both measurements are, the more virulent the pathogen is.

Factors affecting the virulence of bacteria
The virulence of bacteria depends on several factors: These include
• access to the host (exposure)

• adhesion to the host cell
• production of endotoxins and exotoxins leading to damage to host cells and tissues
• evasion of the immune system
• incubation periods.
Access to the host (exposure)

For a bacterial pathogen to cause disease in a host organism such as a person, the person, must first
be exposed to the bacteria. This means that the person must come into contact with the bacteria from
another source. We are exposed to thousands of potential pathogens each day, they are present in the
air we breathe, the food we eat, the objects and surfaces we touch and in the other people and organ-
isms we interact with every day. The vast majority of these potential pathogens will not cause disease.
For a bacterium to cause disease it must first enter the body. There are several places in the human
body where pathogens can enter because cells or bodily fluids are in direct contact with the external
environment. These include:
• mucosal surfaces at the eyes, nose, mouth, anus and urethra

• the skin (especially when it is broken due to cuts or grazes)
• the placenta in pregnant women can act as entry to the unborn foetus.
Adhesion to the host cells
If the bacteria are successful in making entry into the host organism, they must then attach to the cells
of the body tissue. This process of attaching to cells is called adhesion. Bacteria have protein or car-
bohydrate molecules on their cell surface membrane, which they can use to bind to complementary
shaped receptors on body cells. These molecules are called adhesins. Some bacteria also use their
capsules to attach to host cells. They do this by producing a biofilm. A biofilm is formed from a group
of bacteria with slime and capsule layers, this biofilm can then attach to a surface such as a host cell.
Biofilms make it difficult for the bacteria to be removed from the host cell, giving the bacteria time for
the next stage in pathogenicity, invasion. This also protects the bacteria against the immune system
and the action of antibiotics.
Figure 2.7: Biofilms can also form on the surface of teeth causing gum infections (gingivitis) and tooth decay

Production of endotoxins and exotoxins leading to damage to host cells and tissues
Once bacteria have adhered to the host cell, the next step is invasion. This involves the growth and
spread of the bacteria. Bacteria may release toxins as they grow, which will damage the host tissues,
cause disease symptoms and allow the bacteria to evade the immune system. There are two types of
toxin: endotoxins and exotoxins. Endotoxins are lipopolysaccharides, which form part of Gram-neg-
ative bacterial cell walls. When the bacterial cell is destroyed, either by the host’s immune system or
through self-destruction, endotoxins are released.
Endotoxins cause fever (high temperature) in the host organism. A large amount of endotoxin must
be released by the destruction (lysis) of bacteria to cause disease. Examples of bacteria that release
endotoxins include Neisseria gonorrhoea, the bacteria responsible for the sexually transmitted infection
gonorrhoea and Escherichia coli, which causes diarrhoea and vomiting.
Exotoxins are proteins that can be secreted by both Gram-negative and Gram-positive bacteria as they
grow. These toxins then travel around the host organism and interfere with normal cellular function,
they may also help the bacteria to spread around the host organism. Exotoxins do not cause fever but
can trigger an immune response as they are recognised by the immune system. Only a small amount
of exotoxin is needed to cause disease, these types of toxin are described as potent.
Examples of bacteria that release exotoxins include Vibrio cholerae, the bacteria that causes the dis-
ease cholera and Clostridium tetani, which causes the tetanus. Different exotoxins target different
cells, which leads to symptoms and effects that are specific to that disease. The toxin produced by
Clostridium tetani works by inhibiting the release of acetylcholine neurotransmitter at the synapses of
neuromuscular junctions. This results in permanent muscle contraction causing symptoms that begin
as stiffness of the jaw (lockjaw) and then progress to violent muscle spasms in other parts of the body.
As the bacteria reproduce and spread, they produce more exotoxin, which can cause paralysis of the
muscles that control breathing, leading to death. The toxin produced by Vibrio cholerae acts on cells of
the intestine causing these cells to secrete fluid and electrolytes (salts) out of the cell. This causes diar-
rhoea, which can lead to dehydration and death.
Table 2.2: Comparison of endotoxins and exotoxins produced by bacteria
Endotoxins Exotoxins
Lipopolysaccharides Proteins
Part of the bacterial cell wall Secreted by the bacteria
Produced by certain gram-negative bacteria Produced by certain Gram-negative and positive
only bacteria
Released while the bacteria are growing and repro-
Released when the bacteria cell is destroyed
ducing
Not very potent (lots needed to cause disease) Potent (only small amount needed to cause disease)
Cause fever in the host Do not cause fever in the host
Are not recognised by the host immune sys- Are recognised by the host immune system and
tem cause an immune response

Evasion of the immune system
The purpose of the immune response is to detect any substances entering the body that are detect-
ed as non-self. Cells contain protein molecules called antigens on their cell surface membrane. The
immune system can recognise when a foreign antigen is present in the body and trigger an immune
response to get rid of the invader. Bacterial antigens will trigger the immune system, which will lead to
the production of antibodies and phagocytosis of the bacteria to remove the infection. Bacteria can use
strategies to evade the immune system and prevent phagocytosis, giving time to for adhesion to cells
and reproduction.
Strategies for evasion
• Capsule – the bacterial capsule prevents immune cells from attaching to the bacteria and
destroying it by phagocytosis. For example, strains of Streptococcus pneumoniae with a capsule are
more likely to cause infections, such as pneumonia and meningitis, (are more virulent) compared to
the bacterial strains with no capsule.
• Proteases – some bacteria produce and release protease enzymes that protect them from
phagocytosis. Phagocytosis is triggered when antibodies produced by cells of the immune system
bind to proteins on the invading bacteria. Proteases produced by the bacteria bind to and destroy
antibodies and stop them from binding to the bacteria thus protecting the bacteria from
phagocytosis.
• Mycolic acid – the bacterium Mycobacterium tuberculosis, which causes TB, produces a waxy
substance called mycolic acid. This substance protects the bacteria and stops it being broken
down by phagocytes.
• Antigenic variation – some bacteria can change the shape of antigens on their cell surface
membrane. The first time the body is invaded by a particular antigen memory cells are produced,
which remain in the body for many years and can quickly produce an immune response should the
same antigen enter the body again. This means that the immune system can deal with future
infections much more effectively and stop the bacteria before the population has chance to grow.
Some bacteria such as Neisseria gonorrhoeae change the antigens on the cell surface membrane.
This allows secondary infections to evade the host immune system for longer.
Pause point
Discuss why having strategies for evading the immune system increases the virulence of a
bacterial species.
Hint
Recall the steps that take place to create an infection.
Extend
Distinguish between the effect of the production of endotoxins and that of exotoxins.

Factors affecting the virulence of eukaryotes
You will remember that some eukaryotic organisms can also cause disease in humans. For example,
the fungus Candida albicans can cause oral and vaginal thrush infections and fungal meningitis is
caused by various species of the fungus Cryptococcus. Protists are also eukaryotic and some species of
these can be pathogenic. Plasmodium falciparum causes malaria while another protists called Trypano-
soma brucei are responsible for African sleeping sickness. These eukaryotic pathogens also have factors
that affect how virulent they are.
The virulence factors for eukaryotic pathogens include:
• access to the host

• use of adhesins and toxins
• antigenic variation
• the ability to survive inside phagocytic vesicles.
Access to the host

Like bacteria, fungi and protists must gain entry to the host organism before they can start to repro-
duce and cause infection. The host must be exposed to the organism and the organism must bypass
the host’s first line of defence such as the skin, tears and mucus produced by mucosal surfaces.
Use of adhesins and toxins

Like bacteria, fungal pathogens have strategies that allow them to attach to and invade host cells.
Some fungi such as Candida have glycoproteins on their surface, which bond to the phospholipids of
epithelial cells in the skin. These fungi then produce enzymes to break down a structural protein in
epithelial cells called keratin. This helps the fungus to invade the cells. Some fungi can also produce
exotoxins called mycotoxins. Some fungal toxins can have serious consequences in humans. A toxin
called aflatoxin, a potent carcinogen, is produced by Aspergillus fungus. Aflatoxin is a mutagen and
can cause DNA mutations in the cells of the host, leading to the development of cancer in a person
who has inhaled the fungus. Aspergillus also produces a toxin called gliotoxin that causes host cells to
self-destruct. The toxin can cause destruction of phagocytes, allowing the fungus to stay in the body
longer without being destroyed by the immune system.
Protists have unusual, unique mechanisms for attaching to host cells. Giardia lamblia, which causes a
disease of the intestines called giardia, use a disc made of microtubules to attach to the lining of the
intestine. This pathogen causes the intestinal cells to become inflamed, which stops them working as
efficiently to absorb the products of digestion. Plasmodium falciparum produces a protein called eryth-
rocyte membrane protein 1 (PfEMP1), which attaches to the surface of the red blood cells it invades.
This protein causes the red blood cells to stick together and cause blood clots.
Key points
Meningitis – an infection of the meninges membranes that cover the brain and spinal cord.
Epithelial cells – cells of one of the four types of animal tissue. Epithelial tissue lines the cavities
and surfaces of organs and blood vessels.
Keratin – a structural protein that is a component of hair, nails and skin.
Carcinogen – substance capable of causing cancer.
Mutagen – substance that causes changes in the genetics of a cell.
Phagocyte – a type of white blood cell that is capable of engulfing and digesting bacteria and
other small cells.

Antigenic variation
Like bacteria, fungi can evade the immune system using antigenic variation. In antigenic variation the
proteins on the cell surface membrane of the organism that are recognised by the immune system
change from strain to strain. This means that strains of the same fungus or protist may not be recog-
nised by the immune system as a pathogen that has been in the body before as it does not have the
same antigens. The organism with the new antigens will not be destroyed by phagocytosis as quickly as
it would be should it have the same antigens; antibodies will take longer to be produced. The toxin pro-
duced, Plasmodium falciparum, PfEMP1, as described earlier, can be recognised by the host’s immune
system but as the plasmodium replicates over time inside the host, antigenic variation occurs and the
structure of the protein changes. This allows the organism to go unrecognised by the immune system
and cause chronic infection.
The ability to survive inside phagocytic vesicles

Some fungi and protists have strategies that allow them to withstand phagocytosis.
The process of phagocytosis
1 White blood cells cause phagocytes to bind to the antigens on the pathogen’s cell surface
membrane. This can also be helped by antibodies binding to the pathogen’s antigens first.
2 The cell membrane of the phagocyte expands and surrounds the foreign cell.
3 The cell membrane forms a phagocytic vesicle around the pathogen.
4 Lysosomes in the phagocyte release digestive enzymes to digest the phagocytic vesicle thus
destroying the pathogen.
5 Some phagocytes release chemicals called cytokines, which attract new phagocytes to the area
to further target other pathogens present.
A fungus called Cryptococcus causes meningitis and pneumonia and has a capsule made of a polysac-
charide called glucuronoxylomannan. The capsule gives the fungus better resistance towards phago-
cytosis, increasing its virulence. The protist that causes African sleeping sickness, Trypanosoma brucei,
resists phagocytosis by producing a glycoprotein capsule that can also change over time to prevent
recognition by the immune system.
Figure 2.8: The process of phagocytosis

Virulence mechanisms of viruses and prions
Like bacteria and fungi, viruses have factors that determine their virulence and therefore their ability to
cause diseases in humans.
These factors include:
• access to the host

• ability to cause direct cell damage
• latency.
Access to the host
In the first step in viral infection, the virus that must gain access to the host organism and then attach
to host cells. Viruses like all pathogens can enter the body through cavities and mucosal membranes.
Once inside the body, viruses must attach to their target cells to begin the process of replication. Virus-
es are not living and can only replicate inside host cells. As we have seen earlier, viruses have capsids
or envelopes surrounding their genetic material. These structures contain molecules called adhesins.
Adhesins can bind to receptors on the membranes of host cells. This then allows the virus to insert its
genetic material, enzymes and/or other viral proteins that may be needed for the virus to replicate, into
the host cell. Viruses have specific shaped adhesins, which allow them to attach to the target cell they
infect. For example, the protein hemagglutinin is found in the influenza viruses that causes flu. Hemag-
glutinin acts as an adhesin and can bind to glycoprotein receptors on cells of the respiratory system
and intestines. It is only these cells in the body that have the glycoproteins of the correct shape for
hemagglutinin to bind to and so these cells are the host cells of this specific virus. The herpes simplex
virus can cause oral or genital herpes (depending on the strain of virus). These viruses have different
adhesins to those found on the influenza virus. The adhesins present on herpes simplex are known as
glycoproteins gB, gC and gD, they have complementary shapes to bind to receptors called heparan
sulphate, which is found on the cells of the mucosal surfaces of the mouth and genitals.
Figure: 2.9: HIV using adhesin glycoprotein gp120 to attach to CD4 receptors of T cells of the immune
system

Ability to cause direct cell damage
A virus replicates using the host cell organelles. DNA viruses use proteins and enzymes to make their
DNA, which is then used to produce new virus particles through protein synthesis. RNA viruses use
host cell ribosomes to make new viral enzymes and proteins to build virus proteins. Replication of
viruses can cause direct damage to the host cell by causing major biochemical and structural changes.
The changes that happen are referred to as cytopathic – causing cell damage. Cytopathic changes can
cause a host cell to die through bursting (lysis) or through programmed cell death (apoptosis). Both of
these cause the new virus particles to be released into the surrounding fluid, making them free to in-
fect other cells. Rhinovirus works this way, by causing lysis of respiratory cells leading to the symptoms
of the common cold. Some animal viruses leave cells by a process called budding. In budding, the host
cell is not killed immediately but cytopathic changes disrupt the normal functioning of cells, so they are
unable to carry out their normal role.
Key points
Adhesins – molecules on the capsid or envelopes of viruses that are used to attach to receptors
on host cells.
Glycoprotein – proteins with carbohydrate groups attached to the polypeptide chain.
Complementary shape – a relationship between two structures where each structure fits with
the other like a lock and key.
Cytopathic – can cause cell damage.
Lysis – bursting of cells.
Apoptosis – programmed cell death.
Latency
Some viruses have the ability to remain dormant (not active) inside host cells for a period of time. For
example, herpes simplex virus 1 (HSV 1) which causes cold sores can lie latent inside certain nerve
cells. The genome of these viruses can remain latent until activated. Infection with herpes simplex virus
occurs in early life and lifelong latency can occur. This explains why people can suffer from recurrent
cold sores throughout their lifetime. Human Immunodeficiency Virus (HIV) is another example of a vi-
rus that has viral latency, Latent HIV is not affected by the treatments used to slow down infection and
prevent the development of AIDS. When a person is undergoing treatment for HIV, they will never be
completely free of infection. Latent viruses do not trigger an immune response as no new viral particles
are made, this makes It difficult to develop vaccines against latent viral infections.
Prions
There are no specific factors affecting the virulence of prions. This is because prions are not cells but
consist only of misfolded proteins. These proteins can evade destruction by the immune system as
they are not recognised as foreign antigens. The human body may contain these proteins in their
normal form and so the presence of a misfolded protein (prion) can remain undetected and build up
in body tissue. The disease variant Creutzfeldt-Jakob Disease (vCJD) is caused by a mutated prion, a
misfolded form of the protein PrP. When the misfolded PrP protein comes into contact with a correctly
folded form of PrP, it causes the normal PrP to misfold. This process repeats over and over. The num-
ber of prions increases dramatically and can build up in brain tissue causing brain damage and degen-
eration over time.

Evolution and mutation of pathogens
As we have seen, pathogens can evade the immune system by changing their surface antigens. Bac-
teria may also develop more virulent strains through mechanisms of evolution. Bacteria contain small
loops of DNA called plasmids. Plasmids can carry genes that benefit the bacteria in certain situations.
For example, genes that give the bacteria resistance to certain antibiotics can be found on the plasmid.
These beneficial genes arise by chance mutations. Bacteria containing the plasmid with the beneficial
gene can survive the selection pressure (i.e. the presence of the antibiotic). They can then reproduce
and pass on the plasmid to the next generation of bacteria. Bacteria can also copy and transfer plas-
mids to other bacterial cells of the same generation. This is called horizontal gene transfer and can
increase the spread of antibiotic resistance in a bacterial population. Multi-drug resistant tuberculosis
is a form of tuberculosis infection caused by Mycobacterium tuberculosis bacteria that have developed
resistance to the antibiotics used to treat the infection. Mycobacterium tuberculosis does not have the
ability to transfer plasmids by horizontal gene transfer, but instead advantageous chance mutations of
the bacterial genome have been passed from generation to generation. These chance mutations in the
bacterial DNA have caused the cell wall of the bacteria to become a more effective barrier against the
drugs used to treat it and have strains of the bacteria to develop molecular systems that pump drug
molecules out of the bacterial cells. In some TB bacteria, the chance mutations that increase the bacte-
rial virulence can increase the overall mutation rate and allow more mutations to build up, increasing
the chances of further drug resistance occurring.
The virulence of viruses can also be affected by evolution. HIV is one of the fastest evolving viruses
due to its reproduction rate. When HIV reproduces using the body’s T cells, it can accumulate a lot of
mutations. Some of these mutations might increase the virulence of the virus by making it more re-
sistance to treatments but the mutations may also decrease virulence by altering the virus’s ability to
attach to host cells. When a person begins taking HIV medication, it will be effective in controlling the
reproduction of the virus. However, as the virus replicates, chance mutations may occur and cause new
viral particles to be produced that can survive the drugs being taken. Viruses without the advantageous
mutation will not survive the medication taken by the patient. However, viral particles with the muta-
tion can survive, infect more T cells and produce more viral particles. The new viral particles may also
contain the genes to give them resistance to medication. Over time, the medication will stop working
and will need to be changed to prevent the person developing AIDS. This can take anything from a few
weeks, or a few years and the patient will have their condition monitored by professionals. To reduce
the chances of resistance happening, patients are often prescribed several different antiviral drugs at
the same time. This will reduce the chance that viruses with advantageous mutations survive to repro-
duce. The use of “drug cocktails” thus increases the time between HIV infection and the development
of AIDS.
Pause point
Discuss the impact of the evolution of pathogens in causing disease.

Hint
Consider the different methods a pathogen may have for evolving and the impact evolution has
on the host.
Extend
How might scientists try to counteract evolution of pathogens in order to prevent and fight dis-
ease?

A3 Classification strategies
Classification is the process of sorting things into groups based on common characteristics. Classifi-
cation helps us to see connections between groups of organisms and cells with similar properties and
structures and allows us to study evolutionary relationships.
Classifying bacteria
Bacteria share common features that allow them to be classified as prokaryotic cells. However, within
this kingdom, bacteria have other differences allowing them to be classified further.
Shapes
Phenotype refers to the visible characteristics of an organism and includes the shape of the bacterial
cells. In order for bacterial cells to be seen, they are stained and viewed under a microscope. Bacterial
cells can be different shapes and can therefore be classified by their shape.
Table 2.3: Phenotypic shapes of some human pathogenic bacteria
Shape of bacteria Description Examples
Round shaped bacteria can be:

singular: cocci
Streptococcus mutans
Cocci in pairs: diplococci
in chains: streptococci
Staphylococcus aureus
in bunches: staphylococci
Bacillus cereus
Bacilli Rod-shaped bacteria
Escherichia coli
Helicobacter pylori
Spirilla Corkscrew or spiral shaped bacteria
Treponema pallidum
Vibrio Curved rod-shaped or comma-shaped bacteria Vibrio cholerae
Gram staining
Bacteria can also be classified by the structure of their cell wall. Bacteria can be stained by a procedure
called Gram staining. This technique is usually one of the first steps taken in a laboratory to identify
unknown bacteria. Bacteria can be classified as either Gram-positive or Gram-negative. The process
involves staining bacteria so that they can be seen using stains. The first stain, called crystal violet, is
a purple stain t retained by the Gram-positive bacteria as they have thick walls made of peptidogly-
can. These bacteria will appear purple when viewed with a microscope (Figure 2.10). Gram-negative
bacteria have a thinner peptidoglycan wall with an inner and outer membrane, they do not retain the
purple stain. These bacteria cannot be seen
using a light microscope without staining so a
counterstain is used. This is usually a red stain
called safranin. Most species of bacteria can be
classified as either Gram-positive or Gram-neg-
ative, but some bacteria are Gram-variable
or Gram-indeterminate. Some bacteria such
as Mycobacterium tuberculosis do not stain by
either stain during Gram staining and are de-
scribed as acid-fast.
Figure 2.10: Shapes of Gram-positive bacteria:

spirilla, bacilli and cocci

Oxygen requirements
Bacteria can also be classified into groups based on their need for oxygen.
• Facultatively anaerobic bacteria are versatile. They can survive in high or low oxygen
concentrations. The bacteria Staphylococcus aureus, Escherichia coli and Streptococcus spp. are
facultative anaerobes.
• Obligate anaerobic bacteria only grow where there is no or very little oxygen and are killed by the
presence of oxygen. The bacteria Bacteroides spp. and Clostridium spp. are examples of obligate
anaerobes. Species of Bacteroides bacteria living in the colon are obligate anaerobes.
• Obligate aerobe bacteria can only grow where oxygen is in plentiful supply. Mycobacterium
tuberculosis, which causes TB, Is an obligate aerobe.
• Aerotolerant bacteria do not require oxygen but can survive in its presence. The bacteria that
causes tetanus, Clostridium tetani, are aerotolerant.
Figure 2.11: Classification of some bacteria

Classification of viruses
The typical features used to classify organisms do not apply to bacteria as they are not living cells. The
classification of viruses is based on:
• size
• capsid shape
• presence of an envelope
• type of nucleic acid
• mechanism of replication
• host organism (bacteria, fungi, protist, animal or plant)
• pathology (type of disease they cause).
The International Committee on Taxonomy of Viruses (ICTV) began its classification of viruses in the
1970s. More than 200 000 species of viruses have been identified and scientists believe that there are
many more waiting to be discovered.
The Baltimore classification classifies viruses into seven groups (I-VII), depending on their type of
nucleic acid. Viral nucleic acid can exist as DNA and RNA and in the following forms:
• double-stranded DNA with a coding and template strand (ds DNA)

• single-stranded coding strand of DNA (ss -DNA)
• positive-sense single stranded RNA, similar to messenger RNA (mRNA: a copy of the coding strand
of DNA) (ss +RNA)
• negative-sense single stranded RNA complementary to mRNA (ss -RNA)
• double-stranded RNA; one strand is mRNA and the other strand is complementary to it (ds +/- RNA).
Some viruses (Group VII) have gapped nucleic acid. Table 2.4 shows examples of viruses from each
group, their genomes and examples of the diseases caused by these viruses.
Table 2.4: Examples of viruses from each group
Group Genome Examples of viruse Examples of disease caused

Adenoviruses, herpesviruses,
I ds DNA Meningitis, chickenpox, smallpox
poxviruses
II ss +DNA Parvovirus Parvovirus infection
III ss -DNA Reoviruses Gastroenteritis
Hepatitis A, polio, SARs, foot and mouth
IV ss +RNA Picornaviruses, togaviruses
disease, yellow fever, hepatitis C, rubella
Orthomyxoviruses, paramyxovi- Influenza, measles, mumps, Ebola, Mar-

V ss -RNA
ruses, rhabdovirus burg disease, rabies
VI ss +/-RNA Retroviruses HIV/AIDs
Gapped nucleic
VII Hepadnaviruses Hepatitis B
acids

Case study
Giant viruses with cell-like features
In 1957, a scientific paper defined viruses as potentially pathogenic, having only one type of
nucleic acid, DNA or RNA, unable to grow and replicate by binary fission, and lacking their own
metabolic machinery. In 1992, in Bradford, UK, researchers trying to find the organism responsi-
ble for an outbreak of pneumonia found an organism similar to a Gram-positive coccus bacteri-
um. They isolated it from a culture of Acanthamnoeba polyphaga in a water sample from a hospi-
tal cooling tower. The researchers named it Bradfordcoccus, but they found it impossible to get
meaningful results from the usual tests to identify a bacterium.
The organism was stored and in 2003 it was taken to the University of Marseilles, France. In
France, a team used transmission electron microscopy to examine the organism. To their great
surprise, the French researchers found it was an enormous virus and renamed it Acanthamnoeba
polyphaga mimivirus (APMV), in reference to the host organism from which it was first isolated
and because it could mimic a bacterium.
APMV is about the same size as the bacterium Staphylococcus aureus. Since then, other giant
viruses have been discovered and a new viral family, Mimiviridae, has been defined. Mimiviridae
are generally nucleocytoplasmic large DNA viruses.
Inside the capsid of APMV is a lipid membrane and the virus contains fibrils of peptidoglycan. It has
double-stranded DNA and contains 1.2 million base pairs, encoding about 1000 genes, which include
some genes for protein translation apparatus, and enzymes related to DNA repair, RNA modification
and carbohydrate metabolism. These large viruses can be infected by other smaller viruses.
The discovery of these giant viruses has reopened the debate ‘are viruses living organisms?’ In
the future, the entire classification system for living organisms could change and all living organ-
isms on Earth could be placed into one of two groups: ribosome-encoding organisms (cellular or-
ganisms) and capsid-encoding organisms (viral organisms), dispensing with the idea of domains.
Check your knowledge
1 Which aspect of the APMV’s structure was responsible for it being thought of as a
Gram-positive bacterium?
2 In what ways is APMV different from other viruses?
3 How have viruses played a key role in evolution on Earth?
Protists
Protists are difficult to classify into smaller groups because the kingdom is diverse. This means that
different species of protist may not share common features. Protists can differ in their cellular struc-
ture, sources of nutrition and metabolism. Protists may be single-celled or multi-celled. They can also
be free-living, or they may live with other organisms. Protists can act as parasites where the protist
damages the host organism (parasitism) or both organisms can benefit from the symbiotic relation-
ship (mutualism). Many protists are motile, which means they can move around in their watery envi-
ronments. They may use cilia or flagella to move, some protists even have pseudopodia to help them
move. Some protists produce their own nutrition from sunlight (autotrophs) or require a source of
chemicals for their nutrition (heterotrophs).
Pause point
Explain why different characteristics are used to classify different microorganisms.
Hint
Start by listing the similar and different characteristics used to classify the different groups.
Extend
Add examples to your work.

Key points
Parasitism – a type of symbiosis where one organism (the parasite) benefits while the other
organism (the host) is damaged.
Symbiotic – an interaction between two different organisms that live in close association.
Mutualism – a type of symbiosis where both organisms benefit from the association.
Pseudopodia – a temporary bulge from a cell used for movement and feeding.
Autotrophs – an organism that can form nutritional organic substances from simple inorganic
substances.
Heterotrophs – an organism that ingests organic substances for its nutrition.
Assessment activity 2.1 A.P1 A.P2 A.M1
1 Produce a poster of the main groups of microorganisms. For each group include a labelled cell
diagram and functions of the cellular components.
2 Explain how the structures and characteristics of microorganisms are used to classify them.
3 Produce flash cards to explain the key terms used in describing the virulence of a micro-
organism.
4 Explain the structure and function of collagen.
5 Use examples of different diseases to analyse why some microorganisms are more virulent
than others. Try to cover bacterial, viral, fungal and protozoan diseases.

B Examine the transmission and
treatments of infectious disease
Infectious diseases are caused when pathogens enter the body and reproduce causing damage to host
cells. To do this successfully, the pathogen may have strategies to evade the body’s defence systems.
More than 1200 different human infectious diseases are known to date. It is important to be able to
classify diseases into similar groups to allow scientists to study the similarities and differences between
groups of diseases. This helps us to gain a better understanding of the mechanisms of disease, the
spread of disease, prevention measures and possible treatments. The study of infectious diseases and
their spread is known as epidemiology.
B1 Classification overview of infectious disease

Diseases can be classified by:
• target organs
• causative agent
• source.
Target organs
Infectious diseases can also be grouped according to the organs they affect. Pathogens cause damage
to target organs by binding to cells that make up the tissues of that organ.
Some infectious agents target cells that make up the tissues of organs of the intestinal tract. Examples
of intestinal diseases include salmonella, cholera and typhoid fever. Salmonella is caused by Gram-neg-
ative, bacillus bacteria that belong to the Enterobacteriaceae family. Salmonella infection can cause food
poisoning symptoms such as diarrhoea and vomiting. Salmonella infection can be caused by a person
eating food that contains the bacteria. Usually these bacteria are killed by cooking, but ingestion of
poorly cooked meat such as chicken can lead to salmonella infection. Infection occurs when the bacte-
ria reach the small intestine and reproduce in the tissues of the intestine. As they reproduce, the bac-
teria release endotoxins that cause gastroenteritis (diarrhoea and vomiting). Most people can recover
from salmonella infection without treatment from a doctor but young children, the elderly and those
with a weakened immune system may need treatment to prevent dehydration.
Other infectious diseases can target the respiratory tract. The respiratory tract refers to the organs
responsible for breathing. The respiratory system allows us to take in oxygen from the air and expel
carbon dioxide from the body. The respiratory tract consists of the trachea, bronchi, bronchioles, alveo-
li and lungs. The respiratory system structure can be seen in Figure 2.12. Infectious diseases that affect
the sinuses and throat are called upper respiratory infections. Examples of upper respiratory tract
infections include the common cold and laryngitis. Infectious diseases that affect the bronchi, bronchi-
oles, alveoli and lung tissue are called lower respiratory infections. Examples of lower respiratory infec-
tions include bronchitis, pneumonia and tuberculosis. Upper respiratory tract infections usually clear
up by themselves and do not require treatment, but lower respiratory tract infections can be more
serious. Bacterial lower respiratory infections may need antibiotic treatment prescribed by a doctor.

Figure 2.12: The structure of the respiratory system
Infectious diseases can also target the blood stream. The blood stream consists of red blood cells,
white blood cells, platelets and plasma protein suspended in a fluid called plasma. Certain infectious
pathogens can infect the cells of the blood stream.
Examples of blood stream infections are:
• Human Immunodeficiency Virus (HIV)

• Ebola
• Dengue fever
• Malaria.
HIV enters and destroys specific types of white blood cell called macrophages and CD4 T cells.
Ebola is caused by a virus and is found in Central and West Africa, Infection with this virus causes
severe haemorrhaging (blood loss) and is fatal in 60% of infections. The virus infects another type of
white blood cell called dendritic cells. Malaria is caused by eukaryotic organisms such as Plasmodium
falciparum and Plasmodium vivax. Stages in the life cycle of this organism are carried in the saliva of the
female Anopheles mosquito. When a person is bitten by a mosquito carrying the Plasmodium, the par-
asite enters and replicates inside red blood cells. Infected red blood cells are lysed (destroyed) which
reduces the person’s red blood cell count. This can lead to high fever, chills, extreme fatigue and flu-like
symptoms.
The urinary system can also be a target for infectious diseases. The function of the urinary system is to
filter waste products from the blood to produce urine. Urine is produced by the kidneys. Urine is then
stored in the bladder until it is ready to be released from the body through a tube called the urethra.
Urinary tract infections (UTIs) usually occur when bacteria enters the urinary tract but in rare cases
they can be caused by fungi. Lower urinary tract infections, infections of the bladder, are known as cys-
titis. Infection of the upper urinary tract is known as pyelonephritis (kidney infection). The most com-
mon bacterial UTI is caused by Escherichia coli and is more common in women than men. The urethra
that carry urine out of the body is much shorter in women than in men, which means that infectious
occur more easily. Symptoms of UTIs include pain on urination, frequent urination, the feeling of need-
ing to urinate despite an empty bladder and lower back pain. In most cases urinary tract infections can
be treated by a short course of antibiotics. The following factors can increase the chances of develop-
ing a UTI: sexual intercourse, diabetes and obesity.

Some infectious diseases are classified as systemic. This means that they affect the whole body rather
than just one organ, organ system or body part. Systemic infectious diseases usually start off localised
to one particular part of the body but then spread over time to affect the whole body. This can lead
to a wide variety of symptoms. Influenza or “flu” is an example of a systemic infection. Influenza is
caused by an influenza virus. Symptoms include fever and chills, cough, runny nose, blocked nose, sore
throat, headache, earache, tiredness, muscle pain, watering eyes and a rash. Most people can recover
fully from flu with rest and fluids. For those who are vulnerable, such as the young, elderly, immune
compromised or those with underlying illnesses, flu can be more serious. These groups of people are
encouraged to get the flu vaccination each year to reduce their chances of getting flu. Sometimes very
severe flu infections are treated with anti-viral medication.
Pneumonia is a condition that affects the alveoli in the lungs causing them to become inflamed. This
produces symptoms including coughing, chest pain, breathing difficulties and fever. Pneumonia can be
caused by various viruses or bacteria although bacteria are the most common cause. Pneumonia cases
usually clear up within a few weeks, but bacterial pneumonia may require antibiotic treatment. The
elderly or people with other lung conditions may take much longer to recover and may need further
help from doctors. In those who are vulnerable, pneumonia can become systemic and the patient can
develop sepsis. Sepsis (blood poisoning) is life-threatening and occurs when the body responds to
infection by triggering an inflammatory immune response. Sepsis can cause fever, rapid breathing, in-
creased heart rate, confusion, swelling and high blood sugar readings. Sepsis, if left untreated, can lead
to septic shock. Septic shock is characterised by dangerously low blood pressure and abnormal cellular
metabolism. Septic shock can lead to multiple organ dysfunction syndrome (multiple organ failure) and
death occurs in around 50% of cases. Patients with sepsis or septic shock are treated by intravenous
antibiotics and fluids. Sepsis can also be caused by other bacterial infections.
Pause point
Using a diagram of the human body, identify and describe the different target organs for infec-
tious diseases. Give an example of a disease that targets each organ you include.
Hint
Start by reading over the section and summarising the key target organs.
Extend
Improve your work by explaining how infections can become systemic.

Causative agent
Infectious diseases can also be classified by the type of organism that causes the disease. The organ-
ism that causes the disease is also known as the causative agent. Infectious diseases can be bacterial,
viral, protozoan, fungal, prionic, helminthic or ectoparasitic.
Table 2.5: Classification of infectious disease by causative agent

Causative
Description Examples of infectious disease
agent
Cholera, salmonella, staphylococcus infec-
Bacterial Infectious disease caused by bacteria
tions
Viral Infectious disease caused by virus Measles, HIV, influenza
Protozoan Infectious disease caused by protist Malaria, balantidiasis
Athlete’s foot, candidiasis (thrush), aspergil-
Fungal Infectious disease caused by fungi
losis
Variant Creutzfeldt-Jakob disease (vCJD), fatal

Prionic Infectious disease caused by prion
familial insomnia
Infectious disease caused by parasitic

Helminthic Tapeworm, roundworm, whipworm
worm
Infectious disease caused by a parasite Scabies, crab lice (pubic lice), pediculosis
Ectoparasitic
that lives on the surface of the body (head lice)
Figure 2.13: Scanning electron micrograph (SEM) with colour added of prion proteins taken from the brain
of an infected hamster Mag x135 000

Sources of disease
Infectious diseases are spread through the passing of the pathogen from source to host. Once the
pathogen causes disease in the host, the host also becomes a source. The host can pass on the infec-
tious microorganism to others causing the disease to spread.
A disease can be classified as an anthroponosis, zoonosis or sapronosis. The ending of each of these
words, “-nosis” comes from the Greek word for disease.
Anthroponoses (plural of anthroponosis) are diseases that can pass from human to human. This word
comes from the Greek work for man, “anthropos” and the Greek word for disease, “nosos”. Examples
of anthroponotic diseases include smallpox, gonorrhoea and rubella.
Zoonoses (plural of zoonosis) are diseases that can pass from living animals to humans. This word
comes from the Greek word for animal, “zoon” and the Greek word for disease, “noses”. Examples of
zoonotic diseases include rabies, cat scratch disease and yellow fever. Zoonotic diseases rarely cause
extensive outbreaks of disease, but high death rates are usually associated with the presence of dis-
ease.
Sapronoses (plural of sapronosis) are diseases that can pass from the non-living environment to hu-
mans. This word comes from the Greek work for decaying, “sapros” and the Greek word for disease,
“nosos”. Soil, water, decaying plants, dead animals and faeces are environments from which the path-
ogens causing sapronotic diseases can spread to humans. Examples of sapronotic diseases include
legionnaires disease, anthrax and cholera.
Key points
Anthroponosis – diseases that can pass from human to human.

Zoonosis – diseases that can pass from living animals to humans.
Sapronosis – diseases that can pass from the non-living environment to humans.
Case study
Giant viruses with cell-like features
Paul works at a local visitor’s farm. There is a herd of sheep at the farm, Paul must take special
precautions to protect the farm’s visitors from zoonotic infections. One of these infections is
called ovine chlamydiosis. In healthy humans, this infection can cause mild flu-like symptoms,
so visitors are reminded to wash their hands after visiting the farm. This infection can however
be life-threatening to pregnant women and can cause stillbirth or miscarriage. Paul has to make
sure there are signs up to warn pregnant woman of the risks of contact with sheep while they are
at the farm.
1 What is meant by a zoonotic infection?
2 Why is it important that Paul puts up these signs?

B2 Transmission of infectious disease
Transmission of infectious disease refers to the passing of the pathogen from one infected source to
another. This includes the passing of pathogenic bacteria, viruses, fungi, protozoa or prions to individ-
uals who may or may not have had the infection before. The source of the infection, as we have seen
earlier, can be living, e.g. animal, or non-living, e.g. soil. Transmission of disease is by one of two main
methods, direct or indirect.
Direct transmission
Direct transmission is when the pathogen is transferred to an individual through physical contact.
An example of direct contact transmission is the transmission of pathogens during pregnancy, at birth
or through breast feeding. When a foetus (unborn baby) is developing inside the womb, a temporary
organ called the placenta forms. The placenta connects the developing foetus to the uterus using the
umbilical cord. The placenta allows for the foetus to take up oxygen and nutrients required for growth
and allows waste produced by the foetus to enter the mother’s blood stream to be excreted. The pla-
centa is expelled during childbirth after the baby has been born.
Figure 2.14: The placenta provides a surface for exchange of nutrients, oxygen and waste between the de-
veloping foetus and the mother

In 1995 Ford-Jones and Kellner further developed an acronym used to list the most severe infections
that can be passed from mother to child. The acronym is CHEAPTORCHES.
It stands for:
• C – chickenpox and shingles

• H – hepatitis C, D and E
• E – enteroviruses
• A – AIDS (HIV infection)
• P - parvovirus B19
• T – yoxoplasmosis
• O – other (Group B streptococcus, Lyme disease)
• R – Rubella
• C – cytomegalovirus
• H – herpes simplex
• E – everything else sexually transmitted (E.g. chlamydia, gonorrhoea)
• S – syphilis.
If the infection is present in the mother’s blood stream, it can be transmitted across the placenta to the
unborn baby. This can happen in the case of HIV infection. As routine, all pregnant women in the UK
are given the opportunity to be screened for HIV, hepatitis B and syphilis in the early stages of preg-
nancy. If HIV infection is found, anti-viral medication can be given to reduce the replication of the virus
and prevent it being passed from the mother to the developing foetus. If syphilis infection is found, the
mother can begin antibiotic treatment to prevent the bacteria from spreading to the foetus. Infections
can also be spread to the baby during childbirth, as blood from the mother comes into contact with
the baby. Sexually transmitted pathogens such as Neisseria gonorrhoeae bacteria and Herpes simplex
virus can be transferred to the baby as it passes through the birth canal. If it is known that the mother
has either of these active infections, the baby could be delivered using an operation called a caesarean
section to prevent the baby having contact with the pathogens. There are only a small number of infec-
tions that produce enough pathogens in breast milk to cause infection in a breast-feeding baby. These
infections include HIV, chicken pox that developed five days before or two days after childbirth, cyto-
megalovirus and Ebola. In these cases, it is best that the baby is fed formula milk rather than breast
feeding or using expressed milk.
Infections can spread when an infected person has direct physical contact with a non-infected person.
Interactions such as touching, kissing, sexual contact or contact with skin wounds can lead to the trans-
mission of pathogens from one person to another. During this type of transmission, the body fluids
that contain the infectious pathogens come into contact with the body fluids or mucous membranes of
an un-infected person. Some infections can be spread in saliva, e.g., Epstein-Barr virus (glandular fever)
and Streptococcus (sore throat). The most common viruses that can passed by contact with infected
blood are hepatitis B, hepatitis C and HIV. A person can become infected with these viruses if the skin
is broken as they come into contact with infected blood. This could happen if they use a needle that
has infected blood on it (intravenous drug users) or if they accidently injure themselves on an infected
needle (needle-stick injury). There is a lower risk of infection if infected blood comes into contact with
broken skin, eyes, the mouth or nose. Hepatitis B, hepatitis C and HIV can also be spread by unprotect-
ed sexual intercourse between an infected person and an uninfected person. A person with HIV (if not
undergoing effective treatment) will have the virus in some of their bodily fluids. Semen and vaginal
fluids can transmit HIV. The infected fluid must come into contact with a mucous membrane or with
blood for infection to be transmitted. Mucous membranes are found inside the rectum, vagina, penis
and mouth. The risk of transmitting HIV is reduced by using a condom during sexual intercourse. HIV is
not spread through saliva.

Some infectious diseases can be spread from animals to people, we have learnt that these types of
disease are called zoonotic diseases. Some diseases such as rabies can be transmitted directly from
an animal if the infected animal bites or scratches. Rabies virus can be carried by dogs and cats, but in
the UK this is highly unlikely. Rabies is extremely rare in the UK but is found in Asia, Africa and Cen-
tral and South America. Rabies can be transmitted to humans through a bite from an infected animal.
The symptoms of rabies are very similar to those of flu but progress to confusion, anxiety, delirium,
and hallucinations. Rabies is almost always fatal if treatment has not begun before symptoms ap-
pear. There are vaccinations to prevent rabies infection. Some infectious diseases can also be spread
through contact with infected animal faeces. The parasite Toxoplasma gondii can be found in the faeces
of cats and can be present in cat litter. This parasite can be dangerous to pregnant women and those
with a weakened immune system. There is a small risk that the parasite can cause miscarriage or still
birth in pregnant women. If infection occurs in early pregnancy then there is only a small chance that it
can spread to the baby but if it does, the problems that develop are more likely to be serious. If infec-
tion occurs later in the pregnancy, there is more chance of it spreading to the baby, but any conse-
quences are less likely to be severe.
Indirect transmission
Indirect transmission is when the pathogen is transferred to an individual by an intermediate agent
such as the air or an insect.
Some zoonotic infections can be spread when a vector carries the infectious agent. A vector is an
animal that carries the disease-causing agent from an infected individual to an uninfected individual.
A protozoan parasite called Trypanosoma causes Chagas disease and sleeping sickness and can be
transmitted by fleas, ticks and tsetse flies. Lyme disease is the most common tick-borne disease in the
Northern Hemisphere. It is caused by infection with Borrelia burgdorferi bacteria and has the symptoms
of fever and chills, headache, joint pain, muscle pain and extreme fatigue. Lyme disease can usually
be treated effectively with antibiotics but in some people, the symptoms can last for years and disrupt
normal life. The bacteria are transmitted by ticks. Ticks are arachnid parasites that live by feeding on
the blood of mammals. A tick can pick up the bacteria when feeding on infected blood and transmit it
to a new host when feeding on an uninfected individual. One of the tell-tale signs of Lyme disease is a
circular red rash surrounding a tick bite on the skin, this is often described as looking like a bullseye.
Malaria is another disease that is transmitted by a vector. Malaria is serious and sometimes fatal. The
symptoms of malaria are high temperatures (about 38°C), vomiting, diarrhoea and headaches. The
most serious type of malaria is caused by Plasmodium falciparum, infection with this parasite can lead
to the development of breathing difficulties and organ failure, which can be life-threatening. Malaria
can be transmitted by the female Anopheles mosquito from person to person. The mosquito feeds on
blood it obtains by piercing the skin. When the mosquito takes a blood meal from a person with the
parasite in their blood stream, it ingests a form of the parasite called the gametocyte. These gameto-
cytes reproduce in the gut of the mosquito and another form called the sporozoite travels to the mos-
quito’s salivary gland. The Anopheles mosquito will then take another blood meal from another human
and when biting will inject both the parasite and an anticoagulant into the person’s blood stream. The
parasite travels to the person’s liver to grow and multiply before releasing gametocytes into the blood-
stream to infect and destroy red blood cells.
Key points
Fatigue – extreme tiredness.

Arachnid – a class of invertebrate animals having eight legs.
Gametocyte – a cell produced by protozoa that is used for the purposes of reproduction.
Sporozoite – a cell produced in the lifecycle of some parasites that can move.
Anticoagulant – a substance that prevents blood from clotting.

Figure 2.15: The transmission cycle of malaria
Airborne transmission
Some infectious agents can also be transmitted through the air. This type of transmission is referred
to as airborne transmission. The common cold (caused by rhinovirus) and flu (caused by the influenza
virus) are commonly transmitted from person to person by droplets of mucus suspended in the air.
Mucus droplets are expelled into the air when an infected person coughs or sneezes. The mucus drop-
lets can remain suspended for a short period of time and can be breathed in by an uninfected person
in close proximity to the sneeze or cough. The mucus will contain thousands of viral particles which
may then cause infection in the new host. In 2018, the UK government used a campaign to prevent the
spread of flu using the slogan “Catch it, Bin it, Kill it”, which encouraged people to sneeze or cough into
tissues to prevent mucus becoming airborne. Measles is another infection that can be spread by air-
borne droplets and aerosol particles. When an infected person coughs or sneezes, the virus in mucus
droplets or aerosol is released into the air. Measles is highly infectious as a person expels viral particles
in their coughs and sneezes before they develop any symptoms. They could therefore be transmitting
the virus to others while they are not aware they are doing so. Measles symptoms include a cough,
fever, runny nose, sore throat and white spots (Koplik spots) inside the mouth. Most people who have
measles infection will recover fully although there is a higher risk of complications in young children,
pregnant women, people with a weakened immune system and people who are malnourished. About
30% of people with measles will develop complications such as ear infections and bronchitis, but mea-
sles can also cause more serious complications.

Vehicle borne transmission
Infectious agents may also be transmitted non-directly through vehicles. A vehicle is a non-living entity
containing pathogens that can then be picked up by an individual having contact with that vehicle. An
example of a vehicle is a kitchen surface. Salmonella bacteria can be found in uncooked chicken meat.
If this is left on a kitchen surface and the surface is not cleaned effectively, a person could touch the
surface and then ingest the bacteria by hand to mouth action such as eating. The person may then
develop food poisoning, symptoms of which include diarrhoea, vomiting, nausea and stomach cramps.
Door handles, computer keyboards, telephones and handles on shopping trolleys are other common
places where bacteria and viruses can be found. Regular cleaning of surfaces and hands can help to
prevent the spread of infection in this way.
Disease can also be food and waterborne. This means that the infectious agent can be transmitted
through the consumption of infected food or water. There are many different disease-causing patho-
gens that can contaminate food. Usually cooking food thoroughly and ensuring it is consumed before
it is spoilt can prevent enough of the pathogen being present to cause illness. In the earlier example
of salmonella bacteria being present in chicken meat, cooking the chicken fully would ensure that
these bacteria are killed and not transmitted to the person when they consume it. Escherichia coli is
another bacterium that causes “food poisoning”, the symptoms of E. coli infection are severe stomach
cramps, watery diarrhoea and the presence of blood in diarrhoea. There are different strains of E. coli
that cause varying severities of infection. Most people will fully recover from infection but those who
are young, elderly, or vulnerable may develop severe dehydration from diarrhoea, which can be fatal.
E. coli bacteria can be transmitted by inadequately cooked meat, unwashed fruit and vegetables and
unpasteurised milk. Waterborne diseases are caused by pathogens that are transmitted when con-
taminated water is consumed. This can be through drinking the water or washing fruit and vegetables
in contaminated water and then eating them. The bacterium Vibrio cholerae can contaminate drinking
water and cause the disease cholera. Cholera affects around five million people worldwide and is the
cause of tens of thousands of deaths each year in countries in the Global South.

The chain of infection
The chain of infection is the events that must take place to allow pathogens to cause infection in a
person. The chain begins with an infectious agent, the bacteria, virus, fungi, protozoan or prion, that
has the potential to cause infection. The second stage in the chain is the reservoir, a place where the
pathogen can live and reproduce. You have already seen different types of reservoir earlier in the chap-
ter. The next step in the chain is portal of exit. This refers to how the microorganism leaves the reser-
voir. Once the pathogen has left the reservoir using the portal of exit, it must be transmitted. The mode
of transmission, as you have seen, can be direct or indirect. The next step in the chain is the portal of
entry, the route that the pathogen uses to enter the host. The host is the next step in the chain. Once in
the host the pathogen multiply and cause damage to the host cells and tissues. The chain of infection
includes all the necessary features required for infection to happen. It is often useful to think of each
feature as a link in the chain. To stop an infection from happening we try to break at least one of the
links in the chain.
Figure 2.16: The chain of infection

B3 Infectious diseases
This section will cover the causes, signs and symptoms, and progression of a range of infectious dis-
eases. The signs of an infection disease are objective and refer to the physical effects that can be seen
and monitored in different patients with the same infectious disease. The symptoms of the disease
are subjective and relate to how the patient feels, based on their own interpretation of feeling unwell,
examples include headache, nausea and shakiness. The progression of a disease is the change in the
way the infection affects the patient over time.
Bacterial diseases
Meningitis
Meningitis is the term used to describe severe inflammation of the membranes (the meninges) that
protect and cover the brain and spinal cord.
Causes
Meningitis can be caused by a viral or bacterial infection. Bacterial meningitis is rarer but can be very
serious if not found and treated quickly. Different bacteria lead to different forms of meningitis in peo-
ple of different ages. In premature and newborn babies, group B Streptococcus bacteria can cause men-
ingitis. These bacteria, if present in the mother’s vagina, can be passed to the baby during childbirth.
Older children can develop meningitis after infection with Neisseria meningitis or Streptococcus pneumo-
niae bacteria. Another bacterium called Listeria monocytogenes can also cause meningitis in adults.
Signs and symptoms

The symptoms of meningitis include headaches, photophobia (aversion to lights), phonophobia (aver-
sion to sounds), vomiting, muscle and joint pain. If meningitis occurs in babies or young children, they
may not be able to communicate their symptoms and so doctors must look for key signs of infection.
These signs include a high-pitched cry, refusal to feed, becoming stiff, floppy or unresponsive and a
bulging soft spot on the top of the head. Not all of these signs may be present. In older children and
adults signs of meningitis include high temperature, stiff neck (doctors can test this), seizures and spots
or a rash.
If a person is suspected of having meningitis they will be admitted to hospital. A sample of cerebrospi-
nal fluid is usually taken in a procedure called a lumbar puncture. The fluid is checked for bacteria and
viruses. The patient is usually started on antibiotic treatment, often given directly into a vein, before
the test results are returned, this can then be stopped if needed. The patient may also be given steroid
medication to reduce any swelling on the brain.
Progression of disease
The bacteria that cause meningitis usually enter the body in aerosol droplets. These aerosol droplets
are created when an infected person coughs or sneezes, resulting in a non-infected person inhaling
them. The bacteria then bind to receptors on cells in the upper part of the throat before being trans-
ferred to the bloodstream. The bacteria multiply in the bloodstream and the person may start to expe-
rience symptoms. The bacteria eventually end up in cerebrospinal fluid found in the brain and spinal
cord. Most people with meningitis infection will fully recover but it is possible that long-term problems
can arise. Complications range from hearing loss, seizures, memory problems, learning difficulties,
vision loss, kidney problems to loss of limbs. In around 10% of patients, bacterial meningitis is fatal,
the chances of this rapidly increase if treatment does not begin within 24 hours. It is important that if a
person is suspected of having meningitis, that they receive medical attention immediately.

The chances of developing meningitis can be reduced by ensuring you are fully vaccinated against the
common causes. Children receive vaccinations against meningitis as part of the normal vaccination
schedule in the UK. Young teenagers in England are offered a further meningitis vaccine (MenACWY
vaccine) as part of the “teenage booster” vaccine programme in school Years 9 or 10 (UK).
Chlamydia
Cause
Chlamydia is caused by a bacterial infection. It is a sexually transmitted disease that is particularly com-
mon in sexually active teenagers and young adults. The bacteria, Chlamydia trachomatis, can be spread
through sex or contact with infected semen or vaginal fluids.
Signs and symptoms

Many people who are infected with chlamydia do not get any symptoms. In some cases, it might take
several months for symptoms to develop and in other cases, symptoms may disappear even though
there is still an active infection. Symptoms in females include pain when urinating, unusual discharge
from the vagina, pain during sex and bleeding after sex or in-between periods. Males can experience
pain when urinating, a white discharge from the penis and pain in the testicles. As a chlamydia infec-
tion does not usually cause symptoms, it is recommended that those under 25 and sexually active are
tested for chlamydia once a year and when they change sexual partners. Chlamydia infection does not
usually have signs that can be observed by a doctor but there are simple and painless tests for infec-
tion. A swab of the vagina or a sample of urine from a male or female can be sent to be tested for the
presence of the bacteria. The test works by detecting the genetic DNA of Chlamydia trachomatis and
takes around two days to give results.
Treatment of chlamydia infection is a short course of the antibiotic azithromycin or doxycycline. In the
majority of people, this antibiotic treatment will cure the bacterial infection provided that the antibi-
otics are taken correctly and completely. It is important that chlamydia is detected and treated as it
can be spread to other areas of the body and can lead to serious complications. In males, untreated
chlamydia can spread to the testicles and to the tubes that carry sperm from the testicles to the penis.
These tubes, the epididymis, can become painful and inflamed. Inflammation can lead to infertility.
Males can also develop sexually acquired reactive arthritis (SARA) which is a painful inflammatory con-
dition affecting the joints in the body. In females, chlamydia infection can spread to the womb, ovaries
and fallopian tubes. This can cause a serious and painful condition called pelvic inflammatory disease
(PID). PID can lead to further serious complications such as infertility, persistent pain and an increased
risk of ectopic pregnancy.
Figure 2.17: A human Pap smear showing chlamydia infection

Cholera
Cause
Cholera is a bacterial infection caused by Vibrio cholerae. Cholera is not present in the UK but there is a
risk of catching it when travelling abroad, particularly to areas where cholera is epidemic and endem-
ic, typically where access to clean drinking water, treatment and appropriate medical care is limited.
The bacteria are spread by faeces contaminating drinking water and reinfection can occur where waste
contaminates drinking water. This combined with lack of medical care can lead to a fatal cycle of infec-
tion, dehydration and reinfection.
Signs and symptoms

Symptoms of cholera can start from two hours to five days after exposure. In some cases, infections
with certain strains of the bacteria can be asymptomatic (no symptoms) while some strains may cause
minor infections. 5% of infections can be severe and may become life- threatening. The symptoms of
severe cholera are diarrhoea, which is so acute that dehydration can occur within a few hours. The
diarrhoea produced is clear and has a distinctive fishy smell. The infected person may also experience
vomiting and muscle cramps. Dehydration can lead to sunken eyes, wrinkling of the hands and feet
and cold skin. Patients may have deep and difficult breathing, which can be observed by a doctor.
Blood tests can also measure an imbalance of electrolytes (salts) present in the blood. A person with
cholera may also have a rapid pulse and experience drops in their blood pressure.
If untreated, fluid loss can lead to dehydration. Dehydration can cause damage to the nephrons of the
kidneys, which can develop into kidney failure. Severe fluid loss can also cause the blood volume to
decrease causing a drop in blood pressure, collapse of blood vessels and shock. Fluid loss can lead to
imbalances in electrolytes (salts) in the body, which can cause the body pH to become acidic which can
lead to coma and death. Treatment with oral rehydration therapy (ORT) is usually effective at replacing
the fluids lost if enough is given to the patient.
Key points
Epidemic – a rapid increase in the number of new cases of disease in one particular location.
Endemic – cases of the disease are always present in the population of a particular location.
Pause point
Explain why early detection and treatment of bacterial diseases improve the chances of the
patient making a full recovery.
Hint
Use an example of a bacterial disease to describe how the disease progresses over time.
Extend
Explain why there is a higher fatality rate from bacterial diseases in areas with limited access
to clean drinking water and appropriate medical care compared to areas where clean drinking
water, health facilities and appropriate treatment are readily available.

Viral diseases
HIV
Cause
HIV stands for Human Immunodeficiency Virus; it is a virus that infects and damages cells of the im-
mune system. HIV is transmitted through direct contact with body fluids during sexual intercourse with
an infected person or through the sharing of infected needles. People who have unprotected sex with
casual partners or who are intravenous drug users are encouraged to have regular tests for HIV.
Signs and symptoms

Most people who have HIV develop symptoms similar to flu around two to six weeks after infection.
Symptoms include increased temperature (fever), sore throat, tiredness, muscle pain and a rash. Symp-
toms usually last for two weeks but then disappear. An infected person may not experience any further
symptoms for up to 10 years despite having an active viral infection.
Inside the body, the virus binds to receptors on CD4 (T helper) cells and inserts its RNA into the cell.
Once inside the cell, a DNA copy of the RNA is made, which becomes part of the CD4 cellular DNA.
When the cell replicates as part of the normal cell cycle, copies of the viral genome are made. This can
happen immediately or after a period of dormancy. New virus particles are assembled that leave the
infected cell, destroying it in the process. The new viral particles can attach to and infect other CD4
cells. Over time more and more CD4 cells are destroyed and the person’s ability to fight off other in-
fections decreases. A person with HIV will have regular blood tests to measure their CD4 counts. When
the CD4 count falls below 400 per microlitre of blood, the person is diagnosed with AIDS. AIDS stands
for Acquired Immunodeficiency Syndrome and is used to describe the point in HIV infection where the
immune system is no longer able to defend against usually minor infections. Once the immune system
has become damaged, a patient may experience weight loss, night sweats, skin problems and recur-
rent infections. Patients may begin to develop opportunistic infections such as tuberculosis, thrush,
chronic diarrhoea and meningitis. The only measurable sign of HIV is found during a HIV test. Early de-
tection of HIV can allow doctors to start patients on antiretroviral treatment. This treatment can delay
the destruction of CD4 cells and allow infected people to live relatively normal lives for many years.
Ebola
Cause
Ebola is a viral disease that is caused by viruses of the Ebolavirus genus. One of these viral species, Zaire
ebolavirus, is responsible for the largest number of outbreaks in humans. The virus spreads through
direct contact with the blood or bodily fluids of an infected person who has developed symptoms of
the disease. Infection usually occurs through contact with blood, faeces or vomit. Healthcare workers
who care for and treat infected patients are most at risk of infection and must protect themselves by
wearing masks, gowns, gloves and eye protection.
Signs and symptoms

After infection it takes from 2 to 21 days for symptoms appear. These symptoms are influenza- like
symptoms that come on suddenly. Patients experience tiredness, decreased appetite, muscle and joint
pain, headaches and sore throats. Increased body temperature above 38.3°C is a typical sign of Ebola
infection. Symptoms progress to vomiting, diarrhoea, stomach pain and severe dehydration. Five to
seven days after first symptoms, all infected people show some decrease in blood clotting. This can
cause bleeding from mucous membranes, vomiting blood, coughing up blood or blood in faeces. If
bleeding occurs, it indicates that the infection is likely to be fatal. Death, if it occurs, typically happens
between 6 to 16 days after symptoms begin and can be caused by low blood pressure resulting from
loss of fluid.

If an infected person survives, they usually make a full recovery. Sometimes long-term complications
such as muscular pain, hair loss and inflammation of the eyes leading to light sensitivity and vision loss
can occur. The sooner a person receives treatment for Ebola infection, the greater the chance of com-
plete recovery.
Norovirus
Cause
Norovirus is also known as the “winter vomiting bug” and is the most common cause of gastroenteritis.
It is caused by the Norwalk virus, which can be transmitted by indirect contact with infected faeces via
contaminated surfaces, food, water or air. Norovirus is extremely contagious with only a small number
of viral particles required to cause infection.
Signs and symptoms

Between 12 to 48 hours after exposure to the virus, the person experiences diarrhoea, projectile
vomiting, stomach pain and headache. The only other clinical sign in addition to the symptoms experi-
enced by the infected person may be a slightly elevated body temperature. Transmission often occurs
when infected vomit or diarrhoea becomes an aerosol due to flushing the toilet, for example. Infec-
tion can also happen if a person eats or breathes air near to where vomiting or diarrhoea has recently
occurred. It is possible for traces of the virus to be detected a few weeks after a person has recovered
from infection.
Norovirus does not usually require any treatment and clears up within two to three days with the per-
son making a full recovery.
Fungal diseases
Ringworm
Cause
Ringworm is a highly infectious skin infection that despite its name, is actually caused by fungus. There
are different fungi that can cause infection of different parts of the body. Ringworm is caused by a type
of fungi called a dermatophyte that lives on the protein keratin found in the skin. Some examples of
the species of fungi that can cause ringworm include Epidermophyton floccosum, Tinea manuum and
Tinea unguium. Fungal infections can occur on the face (tinea faciei), scalp (tinea capitis), groin (tinea
cruris), foot (tinea pedis) and body (tinea corporis).
Signs and symptoms

Symptoms of ringworm are red rings of scaly, outward growing skin on the affected body parts. Figure
2.17 shows the characteristic ring produced in ringworm infection. The rash rings are itchy and can
spread by scratching the affected area. The most common form of ringworm is an infection of the foot
known as athlete’s foot. Athlete’s foot can be difficult to treat, often requiring long- term use of antifun-
gal creams and sprays. It is called athlete’s foot because those who walk barefoot in public areas, such
as changing rooms at gyms or swimming pools, are at a higher risk of catching the fungal spores that
may have been left behind by an infected person. Athletes are more prone to the infection, as the fungi
grows best in damp areas with poor ventilation such as the footwear of a runner. Symptoms of ath-
lete’s foot includes dry scaly patches on the soles of feet, clusters of blisters on the sides of feet, peel-
ing and moisture between the toes and dry patches on the top of the feet. The signs of ringworm that a
doctor will observe are typically the presence of the characteristic red ring rash.
Ringworm can be treated with antifungal creams applied to the affected skin.

Figure 2.18: The common red circular rash typical in ringworm infection
Candidiasis
Cause
Candidiasis is another infection caused by fungus. The name refers to an infection caused by any type
of a yeast called Candida. This type of infection is most common in the mouth (oral thrush) or vagina
(yeast infection or vaginal thrush), though it can affect other parts of the body such as skin and finger-
nails. In people who are immunocompromised and those who have weakened immune systems, far
more serious infections can occur, potentially leading to severe morbidity and mortality.
Signs and symptoms

Symptoms of candidiasis vary depending on the body part affected but typically the symptoms are
redness, itching and discomfort. In oral thrush a white or cream coloured coating of the tongue may be
observed. Oral thrush is most common in babies less than one month old. This type of infection is not
considered to be a concern unless it persists for more than a few weeks. It can however cause discom-
fort when feeding and the infant will be given a mouth gel to apply to the insides of the mouth and
tongue. Candidiasis infection of the vagina can cause symptoms such as severe itching, burning and
soreness. There may also be an unpleasant discharge. The most causative agent of vaginal thrush is
the organism Candida albicans. It is estimated that 2/3 of females will experience vaginal thrush in their
lifetime. HIV/AIDS, diabetes and repeated treatments with antibiotics increase the chances of vaginal
thrush occurring. In people who are immunocompromised, candidiasis of the oesophagus can occur.
This type of infection has a higher potential for becoming systemic, producing a very serious infection
called candidemia. This can produce mild to extreme flu-like symptoms, pain, chronic tiredness and
other infections. Systemic infection occurs when the yeast enters the bloodstream and spreads to oth-
er organs. Systemic candidiasis is more serious and can lead to sepsis which can be fatal.

Prionic diseases
CJD
Cause
CJD stands for Creutzfeldt-Jakob Disease. It is an extremely rare condition that affects the brain and is
always fatal. CJD is caused by a prion (abnormally folded protein) that causes other proteins to be-
come misfolded. This affects the processes the brain uses to send signals and results in damage to the
neurones (nerve cells). This gives the brain a porous appearance, which is described as spongiform.
Over time the number of misfolded proteins increases causing cell death and loss of function.
Signs and symptoms

The condition is progressive, which means that the symptoms worsen over time. This condition is
always fatal as there is no treatment. Patients are given medication such as steroids, painkillers and
antidepressants to alleviate symptoms. Most people with CJD will die within six months of first symp-
toms although they can live for up to 2.5 years after initial onset. There are different forms of CJD.
• Sporadic CJD (sCJD) is caused by spontaneous misfolding of proteins in a person. This is the most
common form of CJD but still in the UK there are only one or two cases per million people each
year.
• Familial CJD (fCJD) is caused when a mutation is passed from parent to child. This type affects
around one in every nine million people in the UK.
• Acquired CJD (aCJD) or iatrogenic CJD (iCJD) is caused when infection is accidently transmitted
during a medical or surgical procedure. It can also occur if instruments used during brain surgery
are not effectively cleaned before use during a subsequent operation. This type of transmission is
extremely rare as increased awareness has improved sterilisation techniques in surgery.
• Variant CJD (vCJD) is caused mostly by consuming meat from a cow that is infected with the bovine
form of the disease. The form of the disease in cows is called bovine spongiform encephalopathy
(BSE) or “mad-cow” disease for short. Between 1995 and 2000 there was an increase in the number
of cases of vCJD in the UK. This was caused by the practice of using animal remains to feed other
livestock such as cattle. This practice was banned in 1996. There have been no reported cases of
vCJD in anyone born after 1982 and cases of vCJD in people born before this date are extremely
rare. There are now strict rules in place to stop any meat from cows infected or suspected of being
infected from being consumed.

Figure 2.19: Timeline of the vCJD outbreak in the UK
CJD can have a long incubation period with no symptoms for up to 40 years after initial infection. Symp-
toms usually begin with memory loss, impaired judgement and blurred vision. Some people may also
experience an inability to sleep. As the disease progresses symptoms worsen, with people developing
problems with coordination, jerky body movements and blindness. This will eventually develop into the
patient not being able to move or speak and they will fall into a coma. There is no test to confirm di-
agnosis of CJD as this would require a brain biopsy, which is too dangerous to perform while a patient
is alive. Doctors will look for specific signs that indicate CJD. A physical examination can observe prob-
lems with reflexes and muscle spasms and an eye test can detect partial blindness. A lumbar puncture
can be performed where fluid is taken from around the spine. This fluid is tested for a protein, which, if
present, indicates that the person has a high chance of having CJD. A doctor will also observe a patient
for changes similar to dementia that progress quickly.
Pause point
There have been no new vCJD cases in anyone born after 1982. Explain why.
Hint
Consider how vCJS is transmitted and how the disease progresses.
Extend
Discuss why diseases caused by prions are extremely rare.

Parasitic protozoan diseases
Malaria
Cause
Malaria is a tropical disease that is caused by a plasmodium, a single- celled eukaryotic organism. The
most common causative organisms are Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and
Plasmodium falciparum. The female Anopheles mosquito can transfer infective stages of these organ-
isms called sporozoite in their saliva. The sporozoite is transferred to a person’s bloodstream when the
mosquito bites to take a blood meal. The sporozoite travels to the liver where it reproduces to produce
another stage in the plasmodium lifecycle called the merozoite. Merozoites can then infect red blood
cells and reproduce to produce more merozoites. These turn into the male and female gametes of the
organism. When another mosquito comes along and feeds on an infected person, they take up some
of these gametes. The gametes move to the mosquito gut and merge to form immature sporozoites.
Sporozoites then travel to the mosquito salivary glands to develop and can then be passed onto anoth-
er person when the mosquito takes another blood meal.
Signs and symptoms

Symptoms of malaria begin 8 to 25 days after infection. Commonly people experience flu-like symp-
toms such as headaches, fever and vomiting. Infected people may also experience jaundice, blood in
the urine, damage to the eyes and convulsions. There is a typical cycle of sudden coldness followed by
shivering and then fever and sweating. The timing of the cycle varies with the organism causing infec-
tion. For example, P. vivax produces a three-day cycle and P. falciparum produces a 36-48-hour cycle.
Severe malaria is caused by P. falciparum. Severe malaria has the additional symptoms of drowsiness
and weakness. Red blood cells are destroyed, which leads to a decrease in the oxygen transported
around the body. This causes tissues to carry out a lower rate of aerobic respiration producing less
ATP.
If left untreated malaria can progress into a complication known as cerebral malaria. This complica-
tion results in blockages in the blood vessels carrying blood to the brain. This can cause seizures, brain
damage and coma. Other complications can arise from severe malaria infections including liver failure,
shock, a build-up of fluid in the lungs, kidney failure and rupturing of the spleen.
Giardia
Cause
Giardia is an infection caused by a parasitic protozoan organism called Giardia lamblia, the infection is
commonly known as beaver fever. Giardia causes gastrointestinal infection but 10% of infected people
do not experience symptoms.
Signs and symptoms

When symptoms occur, they include diarrhoea, stomach cramps and weight loss. Some people may
also experience vomiting and fever. Infection is characterised by chronic, greasy and foul-smelling diar-
rhoea that can last for several weeks. The long period of diarrhoea can result in weight loss, malnutri-
tion and dehydration. The protozoa are transmitted by contaminated water or food that contain faeces
from an infected person. The faeces will contain Giardia lamblia cysts. A cyst is a dormant stage in the
organism’s life cycle that allows it to exist in hostile environments. An individual may start to see symp-
toms between one to three weeks following transmission of the parasite as the ingested cysts dissolve
and release the organism. The symptoms can last for several weeks but usually clears up on its own. A
patient may be given treatment to prevent dehydration, but people may experience intestinal discom-
fort for some time after infection.

In countries where access to healthcare and treatment is readily available, giardia infection is rarely
fatal. However, in countries where there is a lack of access to clean water supplies, poor healthcare and
a lack of education the infection can have more severe consequences. The main complication that can
arise from giardia infection is dehydration as a result of severe diarrhoea. Children who have giardia
infection can develop malnutrition, which can affect their development and lead to a failure to thrive.
Some people develop lactose intolerance following a giardia infection, which can persist for a long time
after the infection has cleared up.
Helminthic and ectoparasitic diseases

Some infections can also be caused by parasitic worms (helminthic) or by parasites that live on the out-
side of the body (ectoparasites). The table below shows the cause, signs and symptoms and progres-
sion of some of these types of infection.
Table 2.6: The cause, signs and symptoms and progression of common parasitic disease of humans
Name of disease Causative organism Signs and symptoms Progression of disease

Roundworm eggs are ingested and
move into the duodenum. After
1 to 2 weeks the eggs hatch into
larvae. These move into the blood-
stream and move to the lungs. The
Does not usually cause
larvae pass from the lungs to the
any noticeable symp-
throat and are swallowed. The lar-
Caused by nematode toms. Signs of infection
vae then mature into adult worms
Roundworm worm. Most common include presence of
in the intestines and produce
cause of roundworm worms in faeces. Some
more eggs. Adult worms can live
infection is Ascaris people develop a high
for 2 years and produce thou-
lumbricoides. temperature and dry
sands off eggs per day. These eggs
cough during the initial
are released in faeces and can be
larvae stages of infection.
passed onto others through con-
tact with infected faeces. The more
worms present, the more severe
the infection. Severe infections can
lead to blockages in the bowel.
Tapeworm eggs are ingested

The most common through consumption of raw or
Does not usually cause
tapeworms that affect undercooked pork, beef or fish.
any noticeable symp-
humans are: They can also be ingested through
toms. Signs of infection
drinking contaminated water or
Taenia solium (pork include the presence of
by close contact with an infect-
tapeworm) flat, rectangular, pale
ed person. Worms hatch in the
Tapeworm Taenia saginata (beef yellow worms in faeces.
intestine and produce new eggs,
tapeworm) Infection with tapeworm
which are released in faeces.
is rare in the UK but
Hymenolepsis nana Complications can arise if worms
common in other coun-
migrate to other organs such as
Diphyllobothrium la- tries. Symptoms can
the brain or liver. This only hap-
tum (fish tapeworm) include abdominal pain,
pens when worms are caught from
diarrhoea, nausea and
Dipylidium caninum ingesting raw pork and can lead to
vomiting and weight loss.
(dog tapeworm) headaches, seizures, jaundice and
coughing up blood.

Name of disease Causative organism Signs and symptoms Progression of disease
After infestation, lice feed by tak-

ing a blood meal from their host
Infestation of lice. and must stay close to the skin in
Lice are blood feeding order to maintain their tempera-
insects that belong ture. Eggs (nits) are laid by adult
to the Phthiraptera The characteristic symp-
females and take 6 to 9 days to
order. tom of lice infection is
hatch. When eggs hatch, a nymph
itching (pruritus) which
Human pediculosis is released. Nymphs mature into
intensifies 3 to 4 weeks
Pediculosis can be divided into adult lice around 7 days after
after initial infection.
the following: hatching. Adult lice can live up to
30 days as long as they are able
Pediculosis capitis
to feed on the blood several times
(head lice) Lice and eggs can often
a day. Transmission occurs when
be seen in the hair of the
Pediculosis corporis direct contact allows adult lice to
infected part of the body
(body lice) jump to an uninfected person or
Pediculosis pubis where eggs are able to be trans-
(public lice) ferred between individuals. Body
lice can be transmitted by the
presence of eggs on clothing.

B4 Prevention and treatment of infectious diseases
To reduce the chances of catching an infectious disease, there are various preventative measures that
can be taken.
Prevention
Vaccinations
There are vaccinations for some of the more serious infectious diseases and these can either be given
as part of the normal childhood vaccination programme in the UK, to high-risk groups such as the el-
derly or immunocompromised or to those who are travelling to places where there is an increased risk
of disease. The routine childhood vaccination programme in the UK can be seen in Figure 2.20.
Figure 2.20: The routine vaccination programme in the UK as of Autumn 2020

Vaccinations provide immunity to specific diseases through the presence of antibodies. In response to
an infection, a type of white blood cell called B plasma cells produce proteins called antibodies. This
happens naturally when the body detects a non-self-antigen. Antibodies neutralise pathogens or toxins
produced by the pathogen or work by clumping the non-self-cells together, so they cannot attach to
body cells. Vaccinations work either by triggering the body to produce antibodies against a specific
antigen or by providing the person with the necessary antibodies. The antibodies produced are specif-
ic to the antigen that has entered the body. This is why a vaccination against measles would not offer
protection against any other diseases.
Mode of action of vaccines
Active immunity
Vaccinations can work by providing the person with active immunity against a disease. A killed or weak-
ened (attenuated) form of the pathogen may be used to produce this type of vaccination. Vaccines that
contain killed or attenuated pathogen include MMR (measles, mumps and rubella), polio and hepatitis
A. Some vaccines may contain specific pieces of the pathogen that would trigger the immune response
such as a particular protein or capsid. Examples of these vaccinations are the whooping cough, HPV
and pneumonia vaccines. A third type of vaccine that also works by providing active immunity are
vaccines that contain the toxin produced by the microorganism. This toxin will also trigger an immune
response in the person receiving the vaccine. Examples of toxoid vaccines include diphtheria and teta-
nus. All of these vaccine types work by activating the specific immune response that results in the pro-
duction of antibodies and memory cells. These can remain in the body for a long time (in some cases a
lifetime) and provide protection against infection by the actual pathogen. Should the vaccinated person
be infected by the pathogen, the body will be able to produce a faster and greater immune response,
which will allow the infection to be dealt with before it progresses.
Passive immunity
Vaccinations can also work by providing the person with passive immunity against a disease. Antibod-
ies against the pathogen are used to produce the vaccine. These types of vaccine are less common but
include the vaccination against rabies. These types of vaccine produce short- lived immunity and so are
usually given as a preventative measure when travelling to countries where risk of infection is high. In
the case of rabies, the vaccination could also be given following an incident such as a dog bite where
there is a chance that a person has become infected with the virus.
Table 2.7: Comparison of the modes of action of vaccines

Mode of action Vaccine contains Immune response Protection
Dead or weakened path-
Yes – antibodies and
Active immunity ogen, antigenic material Long-lived (often lifelong)
memory cells produced
or toxin
Passive immunity Antibody serum No Short-lived

Antibiotics
Antibiotics are sometimes prescribed to people to prevent infection. They can act as a prophylaxis
against infection. Antibiotics can be used against bacterial infections but do not work against viruses,
fungi or protozoa. Prophylactic antibiotics can be given as a precaution when a patient is having an
operation or has a bite or wound that could get infected. A person with a health condition that means
they have a higher risk of infection may be prescribed antibiotics. A patient who has had spleen remov-
al surgery or is undergoing chemotherapy may prescribed antibiotics to prevent them catching bacte-
rial infections.
Disinfectants
Disinfectants are antimicrobial agents that can be used on non-living objects such as surfaces and
medical instruments. Disinfectants kill microorganisms on the surfaces they are used. Washing with
warm water and detergent may only remove visible dirt but leave microorganisms on the surface.
Cleaning with disinfectants such as phenol, bleach, alcohol, hydrogen peroxide or iodine solution can
kill these microorganisms and prevent them passing to individuals and causing infection. In this way,
disinfectant can prevent the spread of infection. It is important that disinfectants are used in hospitals
and food preparation areas as the risk of pathogens being present on non-living objects is increased.
Key points
Prophylaxis – a measure taken to prevent infection. This could be an action or treatment.

Chemotherapy – type of treatment used in cancer.
Pause point
Distinguish between antibiotics and disinfectants.

Hint
Explain differences and similarities, referring to specific examples.
Extend
Discuss the advantages and disadvantages of using antibiotics and disinfectants to prevent dis-
ease.
Preventative behaviours
Increasing the use of disinfectant when there has been an outbreak of an infection is one behaviour
that can prevent the spread of infection. Other strategies used by hospitals also act to prevent the
spread of infection. Hospitals encourage patients, visitors and staff to use antibacterial hand gel reg-
ularly by placing hand gel on patient beds and at the entrance and exit to hospital wards and depart-
ments. Staff working in hospitals have training on how to prevent the spread of infection and must be
“bare below the elbows”. This means they cannot wear any clothing or jewellery that is on the lower
part of the arms or hands. Hospitals have strict infection prevention strategies that must be followed
before and after medical procedures and surgery.
Outside of hospitals, individuals can also behave in ways to prevent infections from spreading. An
example of a preventative behaviour is regular hand washing especially after going to the toilet and
before preparing or eating food. People should also use tissues when sneezing or coughing and stay
off work or school when they have an infectious illness. Washing raw fruit and vegetables and ensur-
ing that meat is thoroughly cooked before eating can also prevent a person from catching food-borne
infections. Safe sex is a preventative behaviour that can stop the spread of sexually transmitted infec-
tions such as HIV and chlamydia. Safe sex involves using contraception such as a condom for vaginal
penetrative sex, anal penetrative sex and oral sex. Ensuring people have all the vaccines that are part
of the routine vaccination programme as well as having any necessary boosters and travel vaccina-
tions, can help to prevent the spread of infection.

Environmental measures
Precautions can be taken to reduce the spread of infection from the environment. In the UK the mains
water supply is screened and treated to ensure it is safe to drink and use in cooking. This ensures that
any potential water borne pathogens are killed before the water reaches people’s taps. At first the
water is passed through a screen that captures and removes any branches or leaves. Following this, a
solution is added to the water to make impurities such as dirt solidify and float. This is called floccula-
tion. After this the water is filtered twice to remove large and small particles. Sand filters are used to
do this. Some water treatment plants also use carbon and ion exchange processes to remove further
microscopic and dissolved particles from the water. Once the water has been screened and filtered a
small amount of chlorine is added. Less than one milligram of chlorine is added per litre of water. Chlo-
rine acts as a disinfectant and kills any bacteria or fungi present in the water making it safe to drink.
Wastewater from toilets, baths, sinks, washing machines etc. in all homes and industrial settings in the
UK is treated before it enters natural water sources. Many infectious diseases can be spread by the
contamination of water with faeces. Effective treatment of water means that the water can be returned
to the environment with no risk to wildlife or humans. Mosquito nets can be used to prevent the
spread of infections such as malaria that can be transmitted by mosquitoes. A mosquito net is made
of a fine mesh and stops insects from getting through. They are used to cover beds or tents. They can
also be installed on windows or doors. The most effective mosquito nets are those soaked in an insecti-
cide which can reduce malaria cases by up to 70%.
Treatment of infectious disease

If an infection occurs there may be treatments available that can kill the infectious agent or prevent it
from multiplying further. These treatments may eradicate the infection and allow the patient to fully
recover. In the cases of other infections such as CJD, there may be no available treatment to eradicate
the cause and treating the symptoms is the only option.
Antibiotics
Antibiotics can be used to treat bacterial infections. They do not work on infections caused by other
infectious agents. Antibiotics can either be bactericidal, which kill the bacteria or bacteriostatic, which
stop the growth of bacteria. Antibiotics called beta-lactam antibiotics work by disrupting the cell wall
formation in bacteria. Without a cell wall, the bacterial cell bursts. Beta-lactam antibiotics include pen-
icillin and cephalosporin. Macrolide antibiotics work by affecting bacterial ribosomes, stopping them
from carrying out protein synthesis. Blocking this cellular process kills the bacteria as it cannot survive
without protein synthesis. Erythromycin is an example of a macrolide antibiotic. Other antibiotics such
as ciprofloxacin are called quinolones. These antibiotics work by inhibiting the replication of DNA. This
prevents the bacteria from reproducing. Antibiotics can also be classed as broad or narrow spectrum
antibiotics. Broad spectrum antibiotics are effective against a wide range of bacteria, they work against
Gram-positive and Gram-negative bacteria and are often prescribed when the infection has an un-
known cause or is caused by multiple bacteria. Narrow spectrum antibiotics are prescribed less fre-
quently as they only work on certain strains of bacteria.
Antifungals
Antifungal agents can be used to treat fungal infections. Most of these treatments are topical and are
applied to the affected area but some can also be taken orally. Echinocandins are a type of antifungal
that work by disrupting the production of cell walls as the fungi reproduce. An example of this type of
antifungal agent is the treatment for Candida yeast infections. Other antifungal medication works by
preventing the production of new cell membranes when the fungi reproduces. They do this by target-
ing the pathway used to produce a molecule called ergosterol. Ergosterol is a steroid found in the cell
membranes of fungi. Without ergosterol the cell membrane does not work correctly, and molecules
can freely leave the cell, this causes the cell to die.

Antivirals
Antiviral medication can be used to treat viral infections. There are only a small number of antiviral
drugs available, which are specific to certain viral infections. Extensive research has to be carried out to
produce a safe and effective antiviral drug. This is because they replicate inside host cells, so any med-
ication must minimise damage to host cells. The most commonly used antiviral drugs are against HIV,
herpes virus, hepatitis B and C and influenza A and B. Typically viruses follow the same general lifecycle
in order to replicate inside host cells. This cycle can be seen in Figure 2.20. Antiviral medication works
by disrupting one of the stages in the cycle.
Antiviral medication can work by preventing the virus from entering host cells. One way to do this is
to inhibit the production of the antigens on the virus surface so that the viral particles cannot attach
to receptors on the host cells. If a virus cannot enter the host cell, it cannot replicate. Other antiviral
medication works by preventing the replication of the virus inside the host cell or by stopping the suc-
cessful assembly of new viral particles. The synthesis of viral DNA can be inhibited, which prevents the
virus from reproduction. Another mechanism is to stop the assembly of amino acids into the proteins
needed to form the new virus particles.
Figure 2.21: The stages in the lifecycle of a typical virus

Antiprotozoal drugs
Antiprotozoal drugs are those that can be given to treat an infection caused by a protozoan. The
mechanism of action of these drugs varies widely from drug to drug. Malaria is a tropical disease that is
caused by a plasmodium, a single-celled eukaryotic protozoan organism. The most common causative
organisms are Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium falciparum.
Different drugs used to treat malaria work by targeting different stages of the plasmodium lifecycle
and different possible causative agents. Chloroquine works on the blood stage of the infection whereas
primaquine works by killing the merozoites during the part of the lifecycle that happens in the liver.
Doxycycline is another drug that can be given. This drug targets the stage of the plasmodium lifecycle
that occurs in red blood cells of the infected person. This type of malaria treatment is only effective in
Plasmodium falciparum infection. A combination of drugs is often used to treat malaria infections to
ensure that different possible causative species and lifecycles are targeted.
Assessment activity 2.2 B.P3 B.P4 B.M2 AB.D1
1 Choosing one infectious disease that is caused by bacteria, produce a patient information
leaflet that describes how the disease develops. Explain how the patient can protect
themselves against the disease and the possible treatments available.
2 Repeat activity 1 for one viral disease, one fungal disease, one disease caused by a protist
and one prionic disease.
3 Choose one infectious disease that is caused by bacteria and evaluate the treatment
available. Look at the advantages and disadvantages of the treatment and what the benefits
and risks are of having and not having the available treatment.
4 Evaluate the development of new treatments for HIV using links to current issues facing
scientist trying to develop these antiviral drugs.

C Explore the application of techniques
to culture and identify microorganisms
In the laboratory microbiologists will use techniques to grow (culture) various microorganisms and
identify them. This section looks at some of the techniques that may be used and things that scientists
need to consider when working with microorganisms.
C1 Health and safety

When working in an industry including microbiology laboratories, or schools or colleges working with
microorganisms, scientists must ensure they follow the legislation in place to keep workers safe. This is
required by law. The most important piece of legislation that workers have to comply with is the Con-
trol of Substances Hazardous to Health Regulations of 2002. This piece of legislation is often referred to
as COSHH. COSHH includes many substances that are hazardous to health including chemicals but also
biological agents such as microorganisms. To comply with COSHH, workplaces must have procedures
in place for workers to complete risk assessments to prevent or control the potential risk that could
arise. This could include making the correct personal protective equipment (PPE) available and the safe
storage, labelling and disposal of materials and equipment. COSHH also includes the Approved List of
Biological Agents, which classifies microorganisms into one of four hazard groups. Hazard group 1 is
the least hazardous and hazard group 4 is the most hazardous. This list gives workers an indication of
what kind of measures they must put in place for each group of microorganisms. This helps to protect
workers from infection with the microorganism being used in that workplace. It is important that any
workers in the workplace are considered, not just those in direct contact with the microorganism. This
means that safety measures must be put in place for scientists, cleaners, office staff and visitors.
Employers also have a duty under The Health and Safety at Work Act (1974) to inform their employees
about potential hazards and risks in the workplace. Employers also need to consult their employees
on health and safety matters and consider their points of view before making decisions on health and
safety. The Health and Safety at Work Act states that all workplaces must have a risk assessment that
reduces the chances of harm happening and describes procedures that must take place in the unlikely
event that a risk occurs.
Classification of biosafety (levels 1-4)

Biosafety refers to reducing the risk of infection that could arise from working in areas where microor-
ganisms are used. A biosafety level (BSL) is a set of containment precautions used to reduce the risk of
infection in laboratories. A laboratory will decide which level precautions to take based on the organ-
isms they work with and where the microorganism is on the Approved List of Biological Agents. The
lowest level is BSL-1 and the highest is BSL-4. In Table 2.8 you can see the different safety procedures
at the four biosafety levels and the types of microorganism that might be used.

Table 2.8: Safety protocols of the biosafety levels and examples of their use
Example
BSL Risk levels Typical lab safety precedures to follow
microorganisms
B. subtilis, Staphy- This is the type of lab found in schools and colleges.
lacoccus aibus,
Saccharomyces Hand washing on entry to lab and before exit; attention to
Low risk to cerevisiae and personal hygiene; no eating or drinking or mouth pipetting;
individuals non-pathogenic no requirement for containment cabinets,; work can be
1 carried out on open benches; lab has a lockable, but this is
and E.coli
communities (Any organisms not usually locked; people carrying out investigations observe
that do not cause aseptic techniques and wear some protective clothing. such as
disease in healthy disposable aprons; all potentially infectious material has to be
humans) decontaminated before disposal.
Salmonella spp,
Staphylacoccus Pathology and research labs.
Moderate aurea, hepititis B
risk to indi- and C virus, adeno- Procedures of BSLq plus: personnel need more training for
2 viduals and viruses, HIV, path- handling pathogens, given by a senior qualified and competent
low risk to ogenic strains of E scientist; limited access to the lab while work is in progress;
communities coli, Plasmodium extreme precautions with contaminated sharp items; safety
falciparum, Toxo- cabinets used if aerosols will be generated.
plasma gondii
Pathology and research labs dealing with various bacteria,

Mycobacteriuem parasites and viruses that can cause serious or potentially
High risk to tuberculosis, yellow lethal diseases, but for which there are treatments.
individuals fever virus, Yersin-
3 and moder- ia pestis (cause of Procedures of BSL 1 and 2 plus: specific training for lab
ate risk to bubonic plague), personnel for handling pathogens; all procedures for
communities SARS, Chlamydia, handling microorganisms are carried out in containment
rabies virus cabinets; personnel wear protective clothing and may be
required to remove make up or jewellery.
Public health labs and some medical research labs.

Procedures of BSL 1–3 plus: high levels of security for access –
via director; double-door entry with an airlock; airflow systems
Ebola virus, small- and negative pressure in the lab, so air always enters the lab
High risk to pox virus, Herpes and does not flow outwards; air is filtered; multiple contain-
individuals B virus, Marburg ment rooms; lab separate from other buildings; pressurised
4
and commu- virus, Lassa virus, personnel suits; operators manipulate cultures of pathogens
nities Clostridium botuli- by putting their hands inside special gloves inside sealed
num cabinets; established protocols for all procedures; extensive
training for personnel; personnel may be required to shower
before entry and before leaving; own clothes not worn in lab;
protective clothing decontaminated.
Biosafety cabinets
When working with potentially dangerous microorganisms in a laboratory, work is carried out in a bi-
osafety cabinet. The purpose of these cabinets is to protect the worker against infection and to prevent
the accidental release of pathogens into the environment. Biosafety cabinets (BSCs also called laminar
flow cabinets) are enclosed spaces made of stainless steel with no gaps or joints where the microor-
ganism could collect. BSCs are connected to an air supply, which creates a negative air pressure inside

the cabinet. This means that air is always flowing into the cabinet from the outside. The air from the
cabinet is filtered so that no microorganism leaves the cabinet, protecting those in the outside envi-
ronment from infection. BSCs may also have an ultraviolet lamp, which can be switched on to kill any
microorganisms within the cabinet.
Classes of BSCs
There are three classes of BSCs offering increasing levels of protection to the worker and the environ-
ment and increasing levels of protection to the work being carried out.
• Class I: These BSCs provide worker and environmental protection but do not offer any protection
to the work being done. This means that the microorganism being worked with could still become
contaminated by other microorganisms present in the environment, but the worker is unlikely to
become infected if they use the cabinet correctly. Class I BSCs are often used to hold pieces of
equipment such as centrifuges or for procedures that could generate potentially harmful aerosols.
• Class II: These BSCs protect the worker, the environment and the microorganism being worked
with. To protect the work, a fan on top of the cabinet draws sterile air over the work being handled.
This air is then filtered before it leaves the cabinet to protect the worker and environment.
• Class III: These BSCs are sometimes called glove boxes. They are used where maximum
biocontainment is used as they give the maximin protection. The enclosure is airtight and all
material leaves through a double-door autoclave. Workers use gloves attached to the front of the
cabinet to have any contact with the microorganism. This type of cabinet will also have a fan to
draw sterile air over the work and a filter to ensure any air leaving is safe.
Figure 2.22: A biosafety cabinet
Personal protective equipment (PPE)

Personal protective equipment (PPE) refers to the safety equipment that a worker can wear to mini-
mise the risk of exposure to the microorganisms they work with. Different PPE would be worn accord-
ing to the biosafety category being followed. Workers may wear a laboratory coat that fastens to the
neck and has cuffed sleeves. These may either be laundered regularly or may be sterilised after each
use. Scientists may also wear googles or a face shield. They may wear gloves and overshoes if required.
There are some pieces of PPE that are not specific to working with microorganisms but may need to be
worn by the scientist. In the highest level BSL-4, further PPE would be required including full suits or
individual sterile air supply masks. This is because infection with this category of infection could be se-
rious or even lethal. In a school or college laboratory, which would be BSL-1, it is appropriate for teach-
ers and pupils to wear basic protective clothing. This includes laboratory coats or aprons and goggles.
Gloves may also be worn but are not necessarily required.

Pause point
Explain the differences between different levels of biocontainment.
Hint
Include the types of organism in each type of laboratory, specialist equipment they may use and
personal protective equipment that will be worn.
Extend
Discuss the level of training that staff would need in each type of laboratory and the security they
may have.
Methods of sterilisation and disinfection

When working with microorganisms it is important that all equipment used and surfaces worked on
are kept as free as possible from microorganisms. This serves two purposes.
• Protection of the scientists and other personnel in the laboratory from the microorganism they are
working with. This stops people getting infections from the microorganisms they work with.
• Protection of the work from contamination with other microorganisms that may be present in the
environment or on the equipment used. This ensures that any results from the experiment are
valid.
Sterilisation is the process of killing and removing all the microorganisms from a particular location.
This includes the killing/removal of both potentially harmful and harmless microorganisms. Sterilisa-
tion is important, for example, when instruments are used in surgery or in producing canned food. In
the school or college microbiology laboratory equipment has usually been sterilised before it is re-
quired for use. It may be possible to observe sterilisation techniques in a college or school laboratory
used to dispose of contaminated waste or bacterial cultures. Plastic equipment such as Petri dishes
will typically arrive at a school or college in sealed plastic bags that have been sterilised by the manu-
facturer using irradiation. This is where x-rays or gamma rays are used to kill any microorganisms pres-
ent. This makes the Petri dishes sterile and free of microorganisms until the packet is opened. Other
methods including the use of heat, steam and filters to carry out sterilisation can be seen in Table 2.9.
Table 2.9: Examples of sterilisation methods
Sterilisation method Description

Electron beams, x-rays or gamma rays are used to kill and remove microorgan-
Irradiation
isms on disposal equipment.
Flaming –using a flame to heat metal equipment until it glows red.

Incinerating– destroying contaminated waste using high temperatures in an incin-
Heat
erator or furnace.
Tindilisation – boiling in water for 20 minutes in three heating and cooling cycles.
An autoclave is a machine that uses steam heated to 120°C - 135°C. Contaminated
equipment that can withstand this temperature can be sterilised by placing in an
Steam autoclave for at least 15 minutes. Cultures of microorganisms used in school or
college and other BSL-1 laboratories can be placed in an autoclave to be destroyed
before being disposed of in the normal waste.
Liquids that would be damaged by other sterilisation methods can be sterilised
using mechanical filtration. The liquid would be slowly passed through a filter. The
Filtration filter would have pores that allow liquid through but trap microorganisms. Note
that in this method, the microorganism would still need to be killed. Filtering only
separates the microorganism from the liquid.

Disinfection is the use of chemicals on non-living objects such as laboratory benches and glass test
tubes to kill or remove the majority of microorganisms’ present. Disinfection is used when sterilisation
is not possible and is the next best thing to prevent contamination or infection. Different disinfectants
target different microorganisms, so it is important that the correct one is used in different laboratories.
It is also important that the disinfectant is used in accordance with the manufacturer’s guidelines for
diluting with water. This ensures that it works effectively in killing the microorganisms present. Exam-
ples of disinfectants include bleach, hydrogen peroxide and alcohols. Bleach usually contains the active
ingredient sodium hypochlorite, which is effective at killing a wide range of bacteria. Hydrogen perox-
ide works by oxidising compounds that the microorganism needs to survive and reproduce. Alcohols
work by denaturing the proteins in microorganisms causing them to die. Alcohols are widely used as
one method of preventing the spread of microorganisms on hands in hospitals and microbiology lab-
oratories, with hand gel made available for use by staff and visitors. Alcohol wipes may also be used to
disinfect hard surfaces such as benches and chairs in laboratories.
Key points
Sterilisation – the killing or removal of all microorganisms present at a particular location.

Disinfection – the killing or removal of most microorganisms on a physical surface.
Valid – information or a result that can be trusted.
Culture – a method of multiplying microorganisms by providing resources for them to reproduce
in a medium under controlled laboratory conditions.
Aseptic technique
Aseptic technique refers to the practices used in a microbiology laboratory when working with microor-
ganisms. Aseptic techniques are carried out for two purposes.
1 To minimise the risk of infection from the microorganism. This protects scientists working with the
microorganism and any other person who may come in to contact with the microorganism, room
or contaminated waste.
2 To minimise the risk of contamination of the experimental work from microorganisms that may be
present on the scientists or in the laboratory.
When working with microorganisms in a laboratory, other activities being carried out in the immediate
vicinity should be reduced and where possible relocated. For example, if you were working in the class-
room laboratory to produce a streak plate of a bacterial culture, other people should not work on your
bench to complete work such as working on a computer.
When working with microorganisms, you must always thoroughly wash and dry your hands using anti-
bacterial soap and warm water. You must also wear a laboratory coat or apron. You must disinfect the
work surfaces before and after use. When carrying out experiments with microorganisms, you will al-
ways use sterile equipment. Sometimes you may sterilise the equipment yourself using a Bunsen burn-
er, in other cases the equipment will arrive to you sterile and only be used once before it is sent for
re-sterilisation. It is common aseptic technique to always carry out microbiology experiments around a
Bunsen burner flame. A Bunsen burner flame is used to draw air currents upwards, which reduces the
chances that any microorganisms fall onto your work and therefore reduces the risk of contamination.
After you have completed microbiological experiments, any contaminated equipment must be made
safe before it can be disposed of. Metal equipment such as wire loops can be sterilised by holding
them in a hot Bunsen burner flame until the metal glows red. This is called flaming. Other equipment
may be placed in disinfectant. Contaminated Petri dishes or microbiology cultures will be autoclaved
before they are disposed of in normal waste in a sealed bag.

Table 2.10: Step-by-step aseptic technique

Safe culturing of microorganisms
When growing microorganisms in laboratories steps must be in place to ensure that these cultures
remain safe. In school and college laboratories, you will work with non-pathogenic microorganisms.
Although these are safe, culturing could cause them to become pathogenic and so all microorganisms
have to be treated as potentially pathogenic. When growing microorganisms, they must be incubated
(grown at warm temperatures) to ensure they have the energy for adequate growth. Bacterial cultures
should be incubated at temperatures that do not encourage the growth of potential pathogens. This
means that they should be incubated below body temperature. To achieve this, a maximum incubation
temperature of 30°C is used. Aseptic technique should be followed to avoid contamination of cultures.
Contamination could lead to the incubation of human pathogens that have come from the scientist
carrying out the work. Petri dishes should also be sealed with two pieces of tape, one on each side, to
stop the lid from coming off. Petri dishes that have been incubated should never be opened.

C2 Microscopy and staining techniques
Microscopy
There are different types of microscope that can be used to observe microorganisms. Some microor-
ganisms such as fungi, bacteria and protozoa can be seen with a light microscope. Others including
viruses and prions are too small and can only be observed with an electron microscope. In school or
college, you will prepare slides of microorganisms and use a compound light microscope to view them.
Light microscopes are also called optical microscopes, they use visible light to view specimens. Com-
pound light microscopes have an objective lens that magnifies the specimen before a second lens, the
eyepiece lens, causes further magnification to produce an image of the specimen. The person using
the microscope can see the image by looking down the eyepiece lens(es). A light microscope may have
more than one objective lens, which can be rotated into place. These lenses will have different mag-
nifications such as x4, x10 and x40. When each objective lens is used, the total magnification used to
produce the image can be calculated by:
Total magnification = magnification of objective lens x magnification of eyepiece lens
Figure 2.23: Annotated diagram describing how to use a light microscope
The maximum magnification that can be achieved by a light microscope is x1500. As light is used as the
energy source, the wavelength of light limits the maximum resolution that can be achieved. The maxi-
mum resolution of a light microscope is 200nm. This means that if any objects are closer together than
200nm, they will be viewed as one object. Light microscopes can be used to view cells, but intracellular
detail cannot be seen.
Some light microscopes have a special objective lens called an oil immersion lens. This lens allows for
greater magnification than can be achieved by a normal objective lens. In normal objective lenses,
there is air between the objective lens and the specimen. To use this lens, a drop of immersion oil is
placed between the oil immersion lens and the specimen being viewed. The oil has a refractive index
similar to the glass of the lens, so a higher magnification can be achieved.

Key points
Magnification – how many times an image appears compared to the actual specimen.
Resolution – the smallest distance that two separate objects can be apart in a specimen and still
be seen as two separate objects in the image produced. It determines the detail of the image
produced.
Refractive index – the speed of light changes as it passes from one medium to another, for
example from air to glass. This causes the light to change direction, this is known as refraction.
The refractive index of a material is the ratio of the speed light travels in a vacuum to the speed it
travels in that material.
Electron microscopes
Electron microscopes use a beam of electrons to produce very clear, highly magnified images. Elec-
trons have a much shorter wavelength than light and so electron microscopes have a much higher
resolution than light microscopes, typically 400nm. There are two main types of electron microscope
each of which produces different types of image.
Transmission electron microscopes work by passing a beam of electrons through the specimen. They
have a magnification of up to x 50,000, 000 and produce 2D images.
Scanning electron microscopes work by passing a beam of electrons over the surface of the specimen.
They have a magnification of up to x 1, 000,000 – 2, 000, 000 and produce 3D images.
Both types of electron microscope:
• are very large and expensive

• need a lot of skill and training to use
• can only observe very thin specimens
• involves complex and expensive slide production
• require the provision of a vacuum for the electrons to travel through.
Producing slides for light microscopy

In schools and colleges, it is not possible to carry out electron microscopy, but it might be possible to
prepare slides for observation with a light microscope. To view a microorganism, place a small amount
of the culture on a clear, colourless glass slide. This is called mounting. Once mounted on a slide, a cov-
er slip is placed over the top of the specimen to protect it. There are different types of slide that could
be produced.
Flat slides
For most specimens you will use a flat microscope slide. When producing a microbiological slide, it is
important that you follow aseptic technique and use a sterile microscope slide. To observe bacteria, a
heat-fixed smear is produced on a flat microscope slide. A wire loop is used to transfer a loopful of bac-
teria from a bacterial colony into a drop of sterile water on a slide. If a liquid culture is being observed,
then a loopful of the liquid bacterial culture can be added directly onto the slide surface. The slide is
left to dry in air before being heat fixed. Heat fixing is achieved by swiftly passing the slide through the
flame of a Bunsen burner. Heat fixing ensures that the bacteria are not washed off the slide during
staining. All specimens produced for observation by light microscope must be stained in order to be
seen.

Figure 2.24: Use of a Bunsen burner to heat fix a slide
Concave slides
Some species of microorganisms are motile. This means that they move around, this movement can
be observed using a suspension of the living microorganism. This is often used for bacteria, yeast and
protists. This method of slide preparation is known as the hanging drop method.
Step-by-step: Hanging drop method
1 Place a drop of microorganism suspension onto a cover slip using aseptic technique.
2 Place a ring of petroleum jelly around the depression of a concave slide.
3 Position the concave slide over the drop of microorganism suspension
4 Turn the slide and cover slip over so that the suspension is hanging from the cover slip into the
depression of the slide.
5 View the preparation using the oil immersion lens.
Figure 2.25: The hanging drop method, with an oil immersion objective lens, used to view, live motile
specimens

Staining techniques
Slide preparations must be stained in order to be seen. If a specimen is colourless you would not be
able to see anything when you observed it with a light microscope. Different stains work by targeting
different structures within microorganisms. The most common types of staining you will use in schools
and colleges are staining with methylene blue, Grams staining of bacteria and India ink staining.
Simple staining with methylene blue

Once a slide has been heat-fixed, a drop of methylene blue can be added. Excess stain is then washed
off before the slide is viewed with or without oil immersion. Methylene blue binds to and stains the cell
components that have a negative charge. Methylene blue stains the nuclei of cells and can be used to
stain fungi and protozoa. Methylene blue can be used to observe the shape of bacterial cells.
Gram staining
Gram staining is the most widely used stain to stain and differentiate between bacteria. Gram staining
divides bacterial cells into two major groups based on their cell wall structure. These groups are Gram
positive and Gram negative as we have seen previously in the chapter. Gram stain is an example of
differential staining as it allows you to distinguish between two groups of bacteria. Gram staining also
allows you to see the shapes of the bacteria and how they are arranged.
Gram staining involves two different stains. Crystal violet is a purple stain that binds to the thick layer
of peptidoglycan in Gram-positive cell walls. This means that when observed with a light microscope,
Gram-positive bacteria appear purple. The cell walls of Gram-negative bacteria do not retain crystal vio-
let and so they do not appear purple. In order to visualise Gram-negative bacteria, a second stain must
be used. This second stain is called a counter stain. Typically, safranin or fuchsin is used. These stains
cause Gram-negative bacteria to appear red or pink when observed with a light microscope.
Step-by-step: Gram staining
1 Wash your hands with warm water and antimicrobial hand wash.
2 Clean the laboratory bench with antibacterial disinfectants or wipes.
3 Produce a heat-fixed slide of a bacterial culture.
4 Apply the first stain, crystal violet and leave for 30 to 60 seconds.
5 Rinse off the crystal violet by tilting the slide. Use a wash bottle to squirt a gentle stream of
distilled water over the smear.
6 Apply iodine to the smear. Iodine acts as a mordant. It reacts with the crystal violet to form
a complex that traps the stain in Gram-positive cell walls during the next step.
7 Rinse the smear with a 1:1 mixture of acetone and ethanol. Hold the slide at an angle and
rinse for less than 10 seconds.
8 Apply a counterstain such as safranin to the smear. Leave on for 30 to 60 second.
9 Rinse the smear with distilled water by tilting the slide.
10 Dry the slide by allowing it to air dry or by blotting with blotting paper.
11 Observe the slide using a light microscope without and with oil immersion.

Limitations of Grams staining
Grams staining is a good technique because it gives quick results using a relatively simple and inexpen-
sive method. It can be used to determine the best treatment for a bacterial infection. Some antibiotics
specifically target Gram-positive bacteria while some specifically target Gram-negative bacteria. Grams
staining can help narrow down which antibiotics would be effective against a patient’s infection. How-
ever, there are many limitations of the technique. The technique requires the scientist to decide on
the colour that the sample has stained. This can lead to subjectivity as it can be difficult to distinguish
between purple and pink/red. The technique is good because it allows you to identify features that can
help narrow down the identification of the species present. The technique does not, however, allow
identification of the specific bacterial species present. Some bacteria cannot be classified as either
Gram-positive or Gram-negative and so this staining method may not always work.
India ink staining

India ink staining is an example of a negative staining technique. Negative staining is used to study the
morphology (shape), size and arrangement of bacterial cells that are difficult to stain. India ink staining
can be used to stain cells that would be damaged by heat fixing. India ink is an acidic stain that can
donate hydrogen ions and itself become negatively charged in the process. Most bacterial cells are also
negatively charged and will repel the stain. The glass of the slide, however, will not repel the stain and
will appear dark. The bacteria will appear as clear spots against the dark background. When the slide is
observed you can see the shape and arrangement of the cells. Measurements can be made to give an
indication of the size of the cells being viewed.
Step-by-step: India ink staining
1 Wash your hands with warm water and antimicrobial hand wash.
2 Clean the laboratory bench with antibacterial disinfectants or wipes.
3 Using 95% alcohol clean the surface of a glass slide.
4 One the alcohol has completely evaporated, pass the slide through a hot Bunsen burner
flame to sterilise.
5 Place a very small drop of India ink near the end of the slide.
6 Hold an inoculating loop in the centre of a hot Bunsen burner flame and remove a small
amount of the bacterial culture or yeast suspension.
7 Disperse the culture in the stain drop without spreading the stain.
8 Use another sterile slide to spread the drop of stain. Do this by resting the end of the other
slide on the centre of the slide containing the stain drop. Tilt the slide forward towards the
drop forming an angle of less than 90°. Pull the slide towards the drop causing it to spread
along the edge of the spreader slide. Push the spreader slide in the opposite direction to
produce a thin smear of stain.
9 Allow the smear to air dry.
10 Observe the slide using a light microscope without and with oil immersion.
Pause point
Explain the difference between Gram staining and India ink staining.
Hint
Include the types of organism in each type of laboratory, specialist equipment they may use and
personal protective equipment that will be worn.
Extend
Discuss the level of training that staff would need in each type of laboratory and the security they
may have.

C3 Culture of microorganisms
Types of media
When working with microorganisms a substance is needed for them to grow on or in. This is called
the growth media. The growth media used contains all the nutrients that the microorganism needs to
grow. Some types of media are specialised to promote the growth of specific types of microorganism.
Scientists select the growth media depending on the aims of their investigation.
In schools and colleges, people mostly work with bacteria using liquid growth media called nutrient
broth or nutrient agar poured into Petri dishes to produce nutrient agar plates.
Nutrient broths
Nutrient broth is made from beef extract that contains peptones (digested proteins). To make nutrient
broth you would dissolve 8g of beef extract and peptone in sterile water. You would then transfer to
a larger vessel and top up the volume of sterile water to 1L. You would then divide the broth amongst
test tubes stoppered with cotton wool and foil caps or in universal bottles with screw-top lids. You
would then sterilise the broth in an autoclave. Once it has cooled down you can transfer some of the
bacterial culture to the broth using aseptic technique. This is called inoculating. This is done by using a
sterilised wire loop to transfer a drop of liquid solution to the broth or by touching a bacterial colony
with the loop and transferring this to the broth.
When bacteria are grown in broths, they may show characteristic growth patterns. The bacteria could
form a sediment at the bottom of the tube, you would see this as formation at the bottom of the broth.
They could also produce turbid growth throughout the broth solution. This occurs when the bacteria
growing are insoluble. Another growth characteristic in broths is the formation of a pellicle. A pellicle
describes thick growth at the top of the broth tube.
Nutrient agar
Nutrient agar is made by mixing 20g of Bovril (beef extract), 5g of sodium chloride and 15g of agar
powder with 100mL of sterile water. It is important that the agar powder is weighed out in a fume cup-
board to avoid inhalation. Once the ingredients have been mixed together, they form a paste. Further
sterile water is added to make the volume up to 1L. This growth media can also be sterilised in an auto-
clave and allowed to cool and solidify. This can then be melted when required or kept in a thermostati-
cally controlled water bath set to 50°C until you are ready to pour into Petri dishes.

Pouring nutrient agar plates
Petri dishes are supplied in packs of 10. The required number of plates are removed from the package
and laid out on the laboratory bench after it has been disinfected. Care must be taken not to remove
the lid of the Petri dishes until you are ready to pour agar into them. This keeps the inside sterile. When
ready to pour, the lid is lifted off one of the plates, ensuring that the opening is large enough to pour
the nutrient agar in but covering as much of the plate as possible. The lid of the bottle containing the
nutrient agar (if required) is removed, and the neck of the bottle is passed through a hot Bunsen burn-
er flame. The nutrient agar contents are then immediately and smoothly poured into the Petri dish to
cover a depth of about 5mm. The neck of the bottle is re-flamed after pouring and, finally, the lid is
replaced on the Petri dish and the plate is swirled to distribute the liquid agar. This process is repeated
with all the plates required. Once the plates have set, they should be stacked and stored, upside down
in a fridge until you are ready to inoculate them with bacteria or fungi.
Figure 2.26: How to pour an agar plate
When bacteria are grown on solid media, they form colonies. A colony is the result of a single bacte-
rium landing on the solid media and dividing by binary fission. After 24-48 hours, where there was
one bacterium, there will be a visible colony containing several million bacteria that are all genetically
identical to each other and to the bacterium that landed there before incubation. Colonies show typical
growth characteristics. Colony morphology describes the characteristics of an individual colony of bac-
teria growing on the agar. It can be used to help identify the bacteria present. Specific terminology is
used to describe types of colony. This terminology includes:
• Form – the shape of the colony e.g., circular, filamentous.

• Size – the diameter of the colony.
• Elevation – what the colony looks like side on.
• Surface – how does the colony appear e.g., smooth, rough, wrinkled.
• Opacity – is the colony transparent, opaque etc.
• Colour – what colour is the colony e.g., white, cream, red.

Selective media
Microorganisms can also be grown on what is known as selective media. Selective media refers to
any substance that will only allow certain microorganisms to grow. This is different from the ones we
have seen previously (nutrient broths and agars), which are known as general growth media as they
allow a wide range of microorganisms to grow. Selective media supports the growth of one group of
microorganisms while reducing the growth of others. For example, if the only type of sugar in a growth
medium is lactose, then only the bacteria that can utilise lactose (lactose fermenters) will be able to
grow. Selective media is used when you want to isolate specific groups of microorganisms. Another
example is the use of nutrient agar with added antibiotics. If you add penicillin to nutrient agar, this will
promote the growth of Gram-negative bacteria while preventing the growth of Gram-positive bacte-
ria. Only Gram-positive bacteria are susceptible to penicillin. On the other hand, if you want to culture
only Gram-positive bacteria, you could add potassium tellurite or sodium azide to nutrient agar. These
inhibit the growth of Gram-negative bacteria and therefore will allow you to selectively grow only
Gram-positive bacteria. Sometimes growth media is enriched with highly nutritious minerals such as
blood, serum or yeast extract, to encourage the growth of organism that will not grow on other types
of media. Table 2.11 shows some types of specialist agar and their uses.
Table 2.11: Types of specialist agar and their uses
MacConkey agar Mannitol salt agar Blood agar (pH Potato dextrose
Medium
(pH 7.1) (pH 7.4) 7.3) agar (pH 5.6)
Lactose (sugar)
7.5% NaCl
Bile salts
Mannitol (carbohy- Potato extract
Supplementary 5% sheep or horse
Crystal violet drate)
ingredients blood Dextrose (sugar)
Neutral pH red indicator Phenol red (pH indi-
cator)
Peptone
Used for: Culturing Gram-negative Culturing bacteria Culturing fastidi- Culturing fungi in-
bacteria (Gram-positive of the Staphylococ- ous microorgan- cluding yeasts and
growth inhibited by bile cus genus. These isms that require moulds
salts) can withstand high specific nutrients
salt concentrations. to grow. E.g. Neis-
Organisms that ferment
Can differentiate seria gonorrhoeae
lactose appear pink
between pathogenic
Allows scientists
Organisms that do not bacteria such as S.
to detect when
ferment lactose, appear aureus which will
a bacteria may
colourless or cream turn agar yellow
have haemolytic
coloured and non-pathogenic
activity (causes the
(agar stays red)
destruction of red
blood cells)

Differential media
Growth media can also be used to differentiate between different microorganisms that could be
present in a culture or specimen. Table 2.11 shows that MacConkey agar allows you to differentiate
between bacteria that do and do not ferment lactose. Bacteria that can use the sugar lactose as their
energy source, will appear as pink colonies when grown on MacConkey agar. Bacteria that cannot uti-
lise lactose as their energy source will not appear pink.
Starch agar (nutrient agar with added starch) can also be used to differentiate between organisms.
Some bacteria produce and secrete an enzyme called α-amylase which hydrolyses (breaks down)
starch. To carry out the starch hydrolysis test, the bacteria are grown on starch agar and then incubat-
ed. After incubation, iodine solution is added to the agar. Iodine is an indicator that turns blue-black
in the presence of starch indicating that the starch has not been hydrolysed. However, if the bacteria
grown produces α-amylase, the starch will have been hydrolysed. In this case, the iodine will not turn
blue-black.
Methods of cell culture

You will be expected to carry out inoculation of liquid and solid growth media, using aseptic tech-
niques. There are lots of different methods of producing cultures and you may not use all of the ones
described in this section.
Step-by-step: Inoculating liquid media to produce broth cultures
1 Labl your tubes of liquid media (broth) with your name, the date and the microorganism to
be added.
2 Wash your hands with warm water and an antimicrobial hand wash.
3 Ensure the work surface has been disinfected with a suitable disinfectant.
4 Flame a wire loop by holding in the centre of a hot Bunsen burner flame and allow it to cool
(if you are using a disposable, sterile plastic loop, do not flame it).
5 Take the culture tube and loosen the cap. Remove the cap from the culture tube using your
little finger and keep hold of the lid. Do not set it down.
6 Flame the neck of the culture tube.
7 Insert the sterile loop into the culture tube. If it is a liquid culture, obtain a loop-full of culture.
If it is a slant, touch a colony with the edge of the loop. Hold this loop while re-flaming the
neck of the culture bottle and replacing the cap.
8 Pick up the liquid broth tube to be inoculated. Remove the cap (hold onto it) and flame the
neck of the bottle.
9 Insert the loop containing the bacteria into this tube and then withdraw it.
10 Flame the neck of the newly inoculated tube again and replace the cap.
11 Sterilise your wire loop by flaming it or dispose of the loop in a container of disinfect if it is
plastic.
12 Incubate the broth culture produced. It can then be used for further culturing or analysis of
the bacteria.

Stab cultures
Stab cultures are produced by pouring growth media such as nutrient agar into test tubes while it
is molten and then allowing it to solidify. One it is solidified bacteria are introduced into the agar by
stabbing an inoculating needle containing the bacteria into the centre of the agar. The bacteria grow
in the area where the agar has been pierced. Stab cultures are mostly used for short- term transport
of cultures. The bacteria will not grow well so this method is not used for culturing or analysis of the
bacteria.
Slant tubes
Slant tubes, also known as agar slopes, are used for the long-term storage of microorganisms. A slant
tube is produced by pouring molten agar into a tube while it is tilted. As the agar solidifies it forms a
slanted surface on which bacteria and yeast can be made. If the purpose of the slant is to store the cul-
ture until it is needed at a later date, then an agar plug can be created in the top of the tube to prevent
drying out. Slant tubes should be re-cultured once every six to nine months.
Streak plate
Streak plates involve the spreading of bacteria on the surface of solid agar that has been poured into a
Petri dish. This method spreads one loopful of bacterial culture, so that the bacteria on the last streak
are spread out. The aim of a streak plate is to dilute the bacteria culture so that by the end of the
process, a single bacterium lies on the surface of the agar. When the plate is incubated these individual
bacteria will form a colony that is distinct and easy to see. Figure 2.26 shows the procedure for carrying
out a streak plate.
Figure 2.27: How to prepare a streak plate

Pour plates
Another method of culturing bacterium is to use a pour plate. This method also allows analysis of the
effect of substances on the growth of bacteria. To produce a pour plate, 1mL of bacterial culture is add-
ed directly to molten nutrient agar. Before adding the bacteria to the molten agar, it is important that it
has cooled enough not to kill the organism but not so much that the agar solidifies in the tube. Usually
when the agar is at a temperature that is comfortable to touch, it is safe to add bacteria. It is important
to work safely to ensure the agar does not burn you. Once the bacterial culture is added to the molten
agar, the bottle is rolled between the hands to mix it thoroughly. The agar is then poured into a sterile
Petri dish and allowed to solidify. Aseptic technique is observed throughout. Alternatively, the bacterial
culture is first pipetted onto the centre of a sterile Petri dish before cooled molten agar is poured over
the top. When incubated, the bacteria will grow both on the surface and within the agar.
Lawn spreads
Lawn spreads can also be used to culture bacteria. To prepare a lawn spread:
1 Pipette 1mL of bacterial culture into the middle of the solid nutrient agar that has set in a petri
dish.
2 Use a sterilised bent glass rod to spread this evenly over the surface of the agar. One effective way
to sterilise the bent glass rod before and after use is buy dipping in 95% ethanol and setting the
ethanol alight by passing through a hot Bunsen burner flame.
When incubated, lawn spreads produce a lawn of bacteria rather than individual colonies. This type of
plate is useful for investigating the effectiveness of antibiotics or other antibacterial substances.
C4 Quantitative analysis of microbes

There are various ways that microbial growth can be measured quantitatively. One of the factors you
may investigate is a count of the cell population present in a sample. You may carry out total popu-
lation counts in which the total number of cells, living and dead, are counted in a certain volume of a
sample. You can also carry out a viable cell count in which only the number of living cells is counted.
This type of count is useful in giving an indication of the severity of an active infection as it is only living
cells that would cause disease or be transmitted to others.
Yeast cells are large enough to be seen with a light microscope, so a haemocytometer can be used to
count them. A haemocytometer is a special slide that has a grid etched into its middle section, below
the surface of the slide. Figure 2.27 shows a haemocytometer. When a cover slip is placed firmly on the
slide, it forms a chamber of depth 0.1mm so you can calculate the volume of liquid over each etched
square and make a total cell count using the steps below.
• Shake the tube of liquid medium in which the yeast cells have been growing.
• Transfer 1mL of this liquid and add it to 9mL of sterile water in another test tube. This dilutes the
sample by a factor of 10.
• Mix the diluted yeast suspension well and, using a pipette, allow some of the solution to trickle into
the grooves under the cover slip of the haemocytometer.
• Observe the haemocytometer grid under the microscope, using low power first.
Focus on the central grid area where there are 25 squares, each sub-divided into 16 smaller
squares.
• Count the yeast cells in five of the 25 squares (80 small squares): count the central square and each
corner square. The volume of liquid over 80 small squares is 0.02μL.
• If any cells are on the boundaries, only count those on the left and top boundaries and not those on
the bottom and right boundaries.
• Now you know how many yeast cells (n) are in 0.02μL of sample and you can calculate how many
are in 1mL of the undiluted liquid medium.

Number of cells in 1 μL = n / 0.02
So, number of cells in 1 mL of diluted culture = (n / 0.02) x 1000
And the number of cells in 1mL of undiluted culture = (n / 0.02) x 1000 x 10
Figure 2.28: (a) A haemocytometer slide and (b) using a haemocytometer slide to make a total population
count
Key points
Total population count – a quantitative method used to count the number of living and dead
cells in culture.
Viable cell count – a quantitative method used to count the number of living cells in a culture
that are growing and reproducing (viable).
Haemocytometer – a slide with a grid etched into its middle section, used to count cells large
enough to be viewed with a light microscope.

Turbidimetric methods
The amount of growth in a liquid culture can also be measured using a colorimeter, which measures
turbidity. A colorimeter shines a beam of light through a sample that is placed in a special plastic con-
tainer, called a cuvette. A photoelectric cell picks up the light that has passed through the sample and
tells you how much light has been absorbed.
Step-by-step: Measuring turbidity using a colorimeter
1 Fill a cuvette with liquid medium that does not contain any microorganism culture.
2 Set the colorimeter with a blue or green filter.
3 Insert the cuvette into the colorimeter and calibrate the colorimeter by setting the reading to
zero.
4 Fill another cuvette with a sample of the microorganism culture that has yeast or bacterial
cells growing in it.
5 Place the cuvette in the colorimeter using the same filter as before.
6 Measure the absorbance. The greater the absorbance, the greater the growth.
The colorimeter cannot distinguish between particles in the culture medium and cells, which is why
you need to calibrate the colorimeter first so that it “ignores” the particles of the growth medium. You
can also use this method to give a measurement of growth rate by taking samples from the culture at
timed intervals and measuring the absorbance.
Viable counts
Serial dilutions
Serial dilution and subsequence colony counts can be used to give a viable cell count of bacteria. This
gives an estimation of the number of living cells per mL of sample. During a serial dilution the liquid
culture of bacteria is diluted over and over again into a volume of sterile water. Each dilution is then
diluted further by taking a sample of the diluted culture and transferring it to a new volume of sterile
water. This is repeated so that each time the sample is diluted by a factor of 10. Figure 2.28 shows how
the bacteria culture can be diluted using serial dilution.
Figure 2.29: Carrying out a serial dilution

Once the serial dilution has been performed, a small volume of each culture is taken and transferred
to individual nutrient agar plates. Aseptic technique is used to produce a lawn spread of each dilution.
After incubation, a plate with a countable number of colonies is selected. The colonies can be counted
and each one represents a single bacterium. A calculation can then be performed to calculate the num-
ber of viable cells per mL of sample. It is important that any sample volumes and the dilution factor is
taken into consideration.
Step-by-step: Serial dilution and colony counts
1 Label your tubes for serial dilutions and agar plates with your name, the date, the
microorganism to be added and dilution to be produced.
4 Mix the contents of the sample and, using a sterile pipette, withdraw 1mL of the solution.
Hold the lid of the culture bottle after removing and flame the neck of the sample bottle after
removing the lid and before replacing it.
5 Add the sample to a tube containing 9 mL of sterile water. This gives a x 10 dilution factor
(dilution factor of 10-1).
6 Roll the tube between your hand to mix the contents. Transfer 1mL from this tube using a
new sterile pipette and add to another tube containing 9mL of sterile water. This gives
a x 100 dilution factor (dilution factor of 10-2).
a x 1000 dilution factor (dilution factor of 10-3).
a x 10 000 dilution factor (dilution factor of 10-4).
a x 10 000 000 dilution factor (dilution factor of 10-7).
12 Using a sterile pipette each time, transfer 1 mL from the 10-3, 10-4. 10-5. 10-6 and 10-7 dilutions
onto five separate nutrient agar plates and evenly spread the bacteria using a sterile glass
spreader.
13 Secure the plates using two pieces of tape, one on each side, and incubate upside down at
30°C for 24 hours.
14 Examine the plates (do not open them) and select the plate that has between 20 and 200
colonies. Count the colonies. You can divide the plate into sections and use a colony counter
and felt tip pen to spot each colony as you count it. Using this count, you can calculate how
many bacteria were present in 1mL of undiluted broth.

Pause point
Discuss the advantages and disadvantages of colorimetry, hemocytometry and serial dilutions for
measuring the growth of bacteria.
Hint
Think of the pros and cons of each method.
Extend
How can the growth of a multicellular fungus be assessed?
Assessment activity 2.3 C.P5 C.P6 C.M3 C.M4 C.D2

1 Evaluate the development of new treatment for HIV using links to current issues facing
scientists trying to develop these antiviral drugs Carry out Gram staining of a culture of
bacteria and use light microscopy to observe the slide produced. Produce biological drawings
of what you observe and identify as many features and characteristics of the bacteria as you
can.
2 Once you have narrowed down the possible identification of the bacteria, use aseptic
technique to produce the following cell cultures:
a) agar slant
b) streak plate
c) pour plate
d) lawn spread.
3 Compare and contrast the techniques used to identify and cultivate the bacteria. Link your
comparison to the aims of the technique used and the quality of the results obtained.
4 Carry out a total and viable cell count to measure the growth of the microorganism you have
used in activity 2.

D: Investigate the effect of antimicrobial
agents on the growth of microorganisms
You may carry out investigations into the effect of different antimicrobial agents on the growth of
microorganisms. Antibiotics and disinfectants are the two most likely antimicrobials you will investi-
gate When designing an investigation, it is important to plan your method carefully so that the results
you obtain can be trusted. In your experimental design you will need to identify which variable(s) you
change (independent variable), the measurements you will make (dependant variable) and there-
fore the results you will record and the variables you will control (control variables) to ensure results
are valid.
Key points
Antimicrobial – a substance that can destroy or inhibit the growth of microorganisms.

Independent variable – the factor that you change in an experiment.
Dependent variable – the factor that changes as a result of changing the independent variable.
It is the factor that you measure.
Control variables – the factors that you set as constant or aim to keep constant to ensure results
from an experiment are valid and conclusions can be trusted.
D1 Investigating the substances that inhibit the growth of mi-

croorganisms
Designing investigations
You have learnt about different cell culture methods in the previous section. Two of these methods,
lawn spread plates and pour plates, can be used to investigate the effect of antimicrobial agents. Pour
plates are suitable, as they produce an even spread of bacteria within and on the surface of the agar. If
performed correctly, a confluent growth of bacteria occurs. This is where there is a continuous growth
of bacteria covering all parts of the agar. As a result of confluent growth, the number of available bac-
teria for the antimicrobial agent to act on, is controlled. Pour plates can be used when the antimicrobial
being tested is able to diffuse into the agar. A lawn spread also produces confluent growth but only on
the surface of the agar. This method is suitable if the substance being tested does not diffuse into agar
well or if the ability to do so is not known.
A common way to carry out an investigation is to first produce a lawn spread or pour plate of the
bacterial culture being investigated. These plates are then incubated with the antimicrobial agent. The
antimicrobial agent can be added to filter paper discs that are transferred to the surface of the agar
using aseptic technique. Alternatively, wells can be cut into the agar using a sterile cork borer. Con-
trolled volumes of the antimicrobial agent are then added to the wells. In both cases, you would obtain
your results by measuring the zone of inhibition around the antimicrobial agent. When the bacteria
grow confluent growth will be seen across the plate surface. However, where the antimicrobial agent
is effective at inhibiting growth, clear zones will be produced on the agar. These clear zones are called
zones of inhibition. The diameter of the zone of inhibition can be measured using a ruler or digital calli-
per. It is also possible to measure the area of the zones by placing 1mm2 graph paper over the top and
counting the squares.
When investigating the effect of antimicrobials, an important factor to control is the incubation temper-
ature being used. It is important that all bacterial plates being used are incubated at the same temper-
ature as different temperatures may lead to different growth rates and different results.

Case study
Coryn works in a microbiology laboratory and wants to test the effectiveness of different antibi-
otics on a bacterial culture. In order to produce valid results, she must design her investigation
carefully. She decides to produce a lawn spread of the bacterial culture, which will then be incu-
bated with a disc impregnated with the antibiotic. Coryn will measure the diameter of the zone of
inhibition (no growth) around the antibiotic disc. She starts planning her investigation by listing
her variables. She decides that her independent variable is the antibiotic used and her depend-
ent variable is the diameter of the zone of inhibition produced. She identifies two variables to
control; these are volume of bacterial culture added to produce the lawn plate (1 mL) and incuba-
tion temperature (30°C).
1 What equipment might she use to measure the zones of inhibition produced?
2 How might the two control variables identified be controlled?
3 Identify other variables that should also be controlled in this investigation.

Antimicrobial susceptibility tests
Disinfectants are antimicrobial agents that can be applied to non-living objects such as surfaces and
instruments, to kill microorganisms on those surfaces. Examples are phenol, bleach, alcohol, hydrogen
peroxide and iodine solution. Antiseptics are antimicrobial agents that can be applied to skin to reduce
the risk of infection and sepsis. Examples are TCP, hydrogen peroxide, boric acid and salt water. Some
natural compounds such as garlic and essential oils also have antimicrobial properties. Disinfectant,
antiseptics and natural compounds can be tested using antimicrobial susceptibility tests.
Step-by-step: Investigating the antimicrobial effect of disinfectants using a lawn spread
3 Using aseptic technique pour molten agar into a petri dish and allow it to set.
4 Remove the lid of the bacterial culture tube using your little finger and do not put it down. Flame
the neck of the bottle.
5 Using a sterile pipette, extract 1mL of the culture. Do not put the pipette down. Re-flame the neck
of the bottle before replacing the lid.
6 Remove the lid of the Petri dish slightly, keeping it close to the agar. Transfer the bacterial culture
from the pipette to the centre of the agar. Replace the lid. Place the pipette in disinfectant.
7 Dip a glass spreader in 95% ethanol and pass through a Bunsen burner flame to ignite. Allow to
cool.
8 Use the glass spreader to spread the bacterial culture over the surface of the agar.
9 Make up the disinfectants to be tested to their recommended dilutions.
10 Prepare a filter paper disc.
11 Using sterile forceps dip the filter paper disc into the disinfectant and allow to soak.
12 Using sterile forceps place the filter paper disc on the centre of the agar.
13 Secure the plates using two pieces of tape, one on each side and, incubate upside down at 30°C
for 24 hours.
14 Observe the plate and measure any zone of inhibition using a ruler or digital calliper.
Safety tip – ethanol is extremely flammable. Replace the lid of the ethanol dish before passing the glass
spreader through the Bunsen burner flame.
The method above could be modified to investigate the effect of:
• different disinfectants
• different antiseptics
• different natural antimicrobials
• disinfectants vs natural antimicrobials
• different concentrations.
Pause point
Write a method for how you could test the effectiveness of different concentrations of a
disinfectant.
Hint
Start by thinking about which method of cell culture you will use.
Extend
Evaluate the equipment you have chosen and consider how accurate your results would be.

Antibiotics
Antibiotics are chemicals produced by microorganisms that can kill or inhibit the growth of other mi-
croorganisms, mainly bacteria but also some fungi and protozoans. Examples of antibiotics are penicil-
lin, tetracycline and cephalosporins. You can also carry out investigations into the effect of antibiotics
on microbial growth using the lawn spread or pout plate methods.
Antibiotics are generally divided into two groups based on how they work: bactericidal or bacterio-
static. Bactericidal antibiotics work by killing bacteria directly. Some of these work by damaging the
membrane of the bacteria so that the cellular contents leak out. Examples of bactericidal antibiotics
are vancomycin and penicillin. Bacteriostatic antibiotics do not kill bacteria but instead prevent growth
or reproduction of the bacteria. In the longer term this can give the body’s immune system a chance to
remove the bacteria while the population size is not increasing. Tetracycline is an example of a bacteri-
ostatic antibiotic. It works by inhibiting the ribosomes in the bacteria and preventing protein synthesis
from taking place. When carrying out investigations into the effect of antibiotics on microbial growth,
whether the antibiotic is bactericidal or bacteriostatic will affect your results. A larger zone of inhibition
indicates a greater effectiveness at killing bacteria. Bacteriostatic antibiotics may not produce a zone of
inhibition but may cause less of a confluent growth in the area surrounding the antibiotic.
Narrow spectrum antibiotics are designed to target a limited range of bacteria.
You could carry out investigations into the effect of antibiotics on the growth of microorganisms. Your
school/college will be able to buy rings that are impregnated with different antibiotics or individual
discs containing different antibiotics. These can be applied using aseptic technique, onto the surface
of pour plates or lawn spreads. The effectiveness of the antibiotic can be measured by measuring any
zone of inhibition produced. The bigger the zone, the more effective the antibiotic.
Investigations can be carried out to test the effect of:
• different antibiotics
• different types of antibiotic (bacteriostatic, bactericidal, narrow and broad spectrum)
• the age of antibiotics on their effectiveness.

D2 Interpretation, analysis and evaluation of results
To ensure that any results you obtain are as accurate as possible and to allow you to draw valid conclu-
sions it is also important to consider:
• the data collection method used

• the number of repeats you will perform
• any sources of error in the data
• possible limitations in the investigation.
Data collection methods

When measuring the diameter of the zone of clearance you may use a ruler or a digital calliper. A ruler
is typically used to measure in centimetres. When measuring small diameters these units may not be
small enough to show you a clear difference between two zones of inhibition. To avoid this, you may
choose to measure in millimetres instead. Figure 2.29 shows how a ruler can be used to measure the
diameter of a zone of inhibition. Using a ruler, it is only possible to measure to the nearest millimetre.
A measurement made by the ruler has an error of ±0.5mm. This is the maximum uncertainty of the
ruler.
Pour plate of
bacterial culture
Antibiotic disc
Zone of inhibition
ruler
Figure 2.30: Using a ruler to measure the zone of inhibition produced by an antibiotic disc on a pour plate of
a bacterial culture. Measured through the lid of the Petri dish
To improve the accuracy of the measurements made, a digital calliper could be used. Some digital
callipers are able to make measurements to the nearest 0.01mm. A digital calliper, as shown in Figure
2.30, is placed over the lid of the Petri dish. The wheel is then turned to open the prongs of the calliper
so that they represent the size of the diameter of the zone of inhibition. This then gives a digital read-
ing on the screen to the nearest 0.01mm. Measurements made by a digital calliper have less errors due
to the increased sensitivity of the measuring equipment. The maximum uncertainty of a digital calliper
would be 0.005mm.

Figure 2.31: A digital calliper
Repeats and anomalous data

To ensure the data you are collecting is repeatable and can be trusted, you normally take repeat
readings. In an investigation into the effect of antimicrobials on the growth of microorganisms, you
might carry out more repeats on different agar plates or you might divide a plate in half and apply
two discs of antimicrobial. If repeat readings are in good agreement with each other, you can usually
assume that the data is reliable. When you take repeat readings, you can also identify any anomalous
results. An anomalous result is a measurement that does not fit the general trend or pattern. If you
think a result is anomalous, you can disregard it and take a further repeat reading.
Recording and presenting data

When collecting data, it is important that it is presented in a clear and logical way. Usually you will
design a table to collect and display your results. The table should:
• be drawn with ruled pencil lines

• have a complete border around the outside
• have the independent variable in the first column
• have the dependent variable (repeats and mean) in subsequent columns
• have informative headings with units in the columns
• contain measurements recorded to an appropriate level of precision for the measuring equipment
you used.

Example
An investigation was carried out into the effect of three disinfectants on the growth of E.coli bacteria
using the lawn spread method. Measurements of any zones of inhibition produced after 24 hours
incubation at 30°C were made using a ruler in millimetres. The results obtained were recorded in a
correctly drawn table as shown in Table 2.12.
Table 2.12: Effect of different disinfectants on the growth of E. coli bacteria
Diameter of zone of inhibition /mm

Disinfectant
Repeat 1 Repeat 2 Repeat 3 Mean
Bleach 65 63 60 62.7
Hydrogen Peroxide 56 54 57 55.7
Iodine solution 10 60 15 12.5
Table 2.12 has correct headings with units. The table shows that three repeats have been carried out
and all measurements have been recorded to an appropriate level of precision given that a ruler has
been used. Mean values have been calculated. For iodine solution, repeat 2 has not been included
in the mean calculation as it is anomalous. The person carrying out the investigation may choose to
repeat this test.
To present results from an investigation, a graph may be used. Bar charts may be used to display
discrete or categoric data. For the data in Table 2.12, it would be appropriate to draw a bar chart to
display the results.
70
60
Mean diameter of zone of inhibition
50
40
/mm
30
20
10
0
Bleach Hydrogen peroxide Iodine
Disinfectant
Figure 2.32: Bar chart showing the effect of different disinfectants on the growth of E. coli bacteria

Line graphs
Line graph are used when plotting results from continuous data. A line graph produced can result in a
straight line or a smooth curve. As with any graph, the axis must be labelled with informative titles with
units. The scales used should be ascending and have equal intervals. The scale should be chosen so
that the graph area covers as much of the available paper as possible.
90
Mean diameter of zone of inhibition /mm
80
70
60
50
40
30
20
10
0
0 20 40 60 80 100 120
Concentration of ethanol /%
Figure 2.33: Line graph showing the effect of different concentrations of ethanol on the growth of M. luteus
bacteria
The graph in Figure 2.33 includes a line of best fit. The experimental data displayed does not exactly
fit the curve. A line (or curve) of best fit should be drawn so that there are an equal number of points
either side of the line.
Sources of error
Different measuring equipment may measure to different degrees of precision. You have already seen
this when comparing a ruler to a digital calliper.
Percentage error is used in science to give an indication of how error affects the accuracy of the meas-
urements made. To calculate percentage error, you use the following equation:
uncertainty of the measuring cylinder x 100

Percentage error =
measurement made

Worked example
Bobby is investigating the effect of different concentrations of TCP on the growth of S.epidermis
bacteria. He decides to use the lawn spread method and cut wells into the agar. He will then fill
these wells with TCP and incubate the plates for 48 hours at 25°C. He plans to measure the zones
of inhibition produced. Bobby cannot decide whether to use a ruler or a digital calliper to meas-
ure his results. He carries out a preliminary experiment using the recommended dilution of TCP
and makes the following measurements of the zone of inhibition produced:
Ruler/mm = 55
Digital calliper /mm = 54.58
1 What is the maximum percentage error of each piece of equipment?

2 Which piece of equipment would you choose to make the most accurate measurements?
3 What else could affect the accuracy of the measurements made?
Drawing conclusions
Having processed your data using tables and graphs, you should be able to look for trends or patterns
in your results.
You would need to consider:
• Is there a relationship between the variables?

• Do the results support your hypothesis?
• Are your results supported by secondary data?
Before drawing conclusions, you may wish to carry out further statistical analysis of your results. You
may choose to calculate standard deviation. Depending on the type of data obtained you may carry
out statistical tests such as T-tests, correlation analysis or chi-squared tests. You would link your results
and conclusions back to the aims and purpose of the investigation and consider areas where your
results could be improved.
Discussion and evaluation

In science, it is typical to evaluate the investigation carried out by producing a discussion of the results
obtained. in your evaluation, you would explain any possible reasons for anomalous data and suggest
limitations that could reduce the accuracy of any data obtained.

Common limitations you may wish to avoid or include in an evaluation
When carrying out investigations into the effect of antimicrobials, there are some common limitations
that could reduce the accuracy of the results obtained. When planning investigations, you may wish to
tweak your method to avoid some of these. Alternatively, they could offer possible explanations for any
anomalous results.
You may get what looks like zones of inhibition on the agar plate and might assume that this lack of
growth is caused by the antibiotic placed on the agar. It could, however, result from non-standard
confluent growth. This is where the bacteria do not grow evenly across the agar when incubated. Often
this is due to poor technique. You will also need to be aware of any potential contaminating microor-
ganisms that could be competing with the bacteria you are trying to measure. Other bacteria or fungi
that end up on the agar due to poor aseptic technique could outcompete the bacteria being investigat-
ed leading to inaccurate results.
Other possible limitations include:
• use of antibiotic discs that are old or past their recommended use-by date
• antibiotic discs being used that have not been stored at the correct temperature of 4 °C
• poor pouring of agar plates resulting in inconsistent depth of agar in the Petri dish. This would
affect the diffusion of the antibiotic into the agar
• use of incorrect growth conditions for the bacteria.
Assessment activity 2.4 C.P5 C.P6 C.M3 C.M4 C.D2
1 Produce a plan for an investigation into the effects of an antimicrobial on the growth of a
microorganism. Speak to the technicians or teachers in your school or college to find out
what equipment and microorganisms are available.
2 Consider the method you will use to produce the most accurate results and select
appropriate equipment to use.
3 Carry out your investigation using aseptic technique to collect your data.
4 Produce an analysis of your results using the data collected and make valid conclusions.
5 Produce an evaluation of your method, techniques and data collected. Link your conclusions
to the wider impact on prevention and treatment and disease.

Answer to Worked example
Ruler: Percentage error = 0.5 x 100

55 = 0.91%
Digital calliper: Percentage error = 0.005 x100

54.58 = 0.0092%

Think Future Skills
Joanne Metcalfe - Clinical Microbiologist
I work for a company that analyses samples sent from various places including the NHS. I have a
degree in microbiology and have completed masters qualifications in medical microbiology. I have also
completed courses that allow me to practice clinical microbiology and to become a registered clinical
microbiologist. The company I work for monitors and analyses microbial cultures and samples using
computer software and a range of practical methods. Typical tasks I might complete include:
Identifying infections in patient samples using a variety of microbiological, biochemical and molecular
methods
Carrying out tests to assess the virulence of different microorganisms
Carrying out research to prevent the spread of infectious microorganisms in hospitals and other
clinical settings
Recording, analysing and interpreting scientific data
Writing reports
Communicating with healthcare professionals or clinical colleagues
Focusing your skills

I need the following skills to do my job competently:
• enthusiasm about microbiology

• multi-tasking
• quick learner
• problem solving
• good communication skills
• sound technical judgement
• attention to detail
• ability to be organised and meet deadlines
• analytical skills
• high emotional intelligence
• desire to commit to professional development and improvement.

Getting ready for assessment
Hannah is studying for a BTEC Extended Certificate in Applied Human Biology. She was given an assign-
ment as part of her practical portfolio. She was asked to investigate the effect of garlic, as an antimicro-
bial, on the growth of bacteria.
How I got started

I gathered all of my notes on antimicrobial agents and techniques used to investigate the effect of an-
timicrobials on the growth of bacteria. I used relevant and appropriate sources to find out more about
the antimicrobial properties of garlic. I found a reference to an article in the magazine Scientific Amer-
ican and the technicians at my college were able to help me find the article. I also found some useful,
peer reviewed websites via the internet.
How I brought it all together

I decided to take an extract of garlic and then use the well method on a lawn spread. I first used a bac-
terial slant that my college had brought in and inoculated nutrient broth to make a liquid culture. After
incubating this culture for 48 hours at 25°C, I used it to produce a lawn spread of bacteria on nutrient
agar plates. Using a cork borer, I made a well in the centre of each plate. Then, I added a known volume
of a known concentration of garlic extract and incubated the plates for 24 hours at 30°C. I repeated
this for all of my different concentrations of garlic on separate lawn spreads. I also carried out three
repeats of each concentration on different lawn spreads. This would allow me to see if my data was
reliable and carry out a statistical test. I got my results by using a digital calliper to measure any zones
of inhibition that were produced around the wells where I added the garlic. I recorded my results in a
scientific table.
What I learnt from the experience

I learnt that garlic has antimicrobial properties. I found that some concentrations of garlic produced
larger zones of inhibition and therefore are more effective at reducing bacterial growth.
I could have extended the investigation by trying other extracts, for example tea tree oil.
Think about it
1 Have you made a plan with timings so you can complete your assignment by the agreed sub
mission date?
2 Have you arranged to meet with the science technicians or your tutor to discuss what re
sources are available for your work?
3 Have you researched all the background information about the bacteria and the chemicals
you will use?
4 Have you made notes of the sources of your information so you can reference all sources
properly?
5 Have you considered all the safety aspects of your investigation and practised using aseptic
techniques?


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BTEC Level 3 National

Text © Pearson Education Limited

Designed by Peter Stratton

Typeset by Peter Stratton

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Cover photo/illustration © Pearson Education Ltd

First published 2021

Publication code VQ000185

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A note from the development team

Practical microbiology and infectious diseases 2

Practical microbiology and infectious diseases 3

Features of this document

• explain what your learning is about

Features that explain what your learning is about

Getting to know your unit

Features that help you build your knowledge

Practical microbiology and infectious diseases 4

Features to help you reflect on and evaluate your learning

Think future skills

Practical microbiology and infectious diseases 5

How you will be assessed

Learning aim B: Examine the transmission and treatments of infectious

Practical microbiology and infectious diseases 6

Practical microbiology and infectious diseases 7

A Understand the classification and

• Animals – these cells are eukaryotic with no cell wall

Practical microbiology and infectious diseases 8

Figure 2.1: Generalised structure of a bacterial cell

The cell wall

Practical microbiology and infectious diseases 9

Figure 2.2: Bacterial reproduction by binary fission

Practical microbiology and infectious diseases 10

Figure 2.3: Typical bacterial growth curve

Ribosome – organelle responsible for protein synthesis.

Practical microbiology and infectious diseases 11

• Can be single-celled such as yeast or multi-cellular such as mushrooms.

Practical microbiology and infectious diseases 12

1 The parent cell forms a bud.

Distinguish between prokaryotic and eukaryotic cells.

Practical microbiology and infectious diseases 13

• Consist of a capsid (protein coat) made of smaller units, called capsomeres.

Practical microbiology and infectious diseases 14

Practical microbiology and infectious diseases 15

Pathogen – an infectious microorganism or agent that can cause disease.

Avirulent Increasing virulence Highly

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• access to the host (exposure)

Access to the host (exposure)

• mucosal surfaces at the eyes, nose, mouth, anus and urethra

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Table 2.2: Comparison of endotoxins and exotoxins produced by bacteria

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• access to the host

Access to the host

Use of adhesins and toxins

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The ability to survive inside phagocytic vesicles