Module 5 Microbiology For Pharmacy

Level 3 in
Pharmaceutical Science
MODULE 5 MICROBIOLOGY FOR PHARMACY
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Level 3 Pharmaceutical Science
Module 5 – Microbiology for Pharmacy
Contents
Introduction ..................................................................................................................... 2
Chapter 1 - Types of Micro-organisms ............................................................................... 3
1.1 Bacteria ...................................................................................................................... 3
1.2 Fungi .......................................................................................................................... 6
1.3 Protozoa ..................................................................................................................... 7
1.4 Viruses ....................................................................................................................... 8
Chapter 2 - Looking at Micro-organisms .......................................................................... 13
2.1 Light Microscope ...................................................................................................... 13
2.2 Electron Microscope ................................................................................................ 13
2.3 Other Forms of Microscopy ...................................................................................... 14
Chapter 3 - Growth of Micro-organisms .......................................................................... 17
3.1 Bacterial Counting .................................................................................................... 17
3.2 Growth Curves ......................................................................................................... 20
3.3 Environmental factors and growth ........................................................................... 21
3.4 Laboratory Media ..................................................................................................... 23
Chapter 4 - Microbial Control of the Environment ........................................................... 26
4.1 Environmental Control ............................................................................................. 27
4.2 Sampling Techniques ................................................................................................ 30
4.3 Disinfectants and Antiseptics ................................................................................... 31
4.4 Sterilisation .............................................................................................................. 34
4.5 Cross-Contamination ................................................................................................ 34
Chapter 5 - Infectious Diseases ........................................................................................ 38
5.1 Cause and Transmission of Infection ........................................................................ 38
5.2 Pathogenic Micro-organisms .................................................................................... 40
Copyright Buttercups Training Ltd – October 2019

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Introduction
Microbiology is the study of very small living organisms - so small, they cannot be seen
without a microscope. Even though we cannot see them, micro-organisms are all around us.
We are probably most aware of them when they result in problems, like causing food to go
off or when a cut becomes infected. However, most micro-organisms are beneficial or
harmless to us. For example, the 'rotting' of the compost heap, essential for soil
fertilisation, is due to micro-organisms. Micro-organisms are also used in making foods,
such as bread, wine and pickles.
Two important terms that you need to be aware of in this module are eukaryotic and
prokaryotic and they are used to classify different types of cells.
A eukaryotic cell includes animal, plant, fungi and some protozoan cells. It describes a cell
which has a nucleus and therefore its genetic information (DNA) is surrounded by a nuclear
membrane.
A prokaryotic cell includes bacteria and archaea (although you do not need to learn about
archaea on this course). They are different from the eukaryotic cells as their genetic
information is not surrounded by a membrane, and therefore they do not have a nucleus.
They instead have a nucleoid (ring of circular DNA) which is located loosely in the cytoplasm.
Micro-organisms will either reproduce themselves through asexual or sexual reproduction

and you will be coming across references to these in the next chapter, so it is worth touching
on them now.
Sexual reproduction occurs when the genetic nuclei of two different cells combines and is
brought together to form a new cell with a mixture of both the parents’ genetic information.
Sexual reproduction is what occurs when a sperm and egg come together to produce a
zygote (this later develops into an embryo)
Asexual reproduction occurs when a cell simply divides in two and the genetic information
of the new cell is identical to that of the parent cell. In essence, a clone has been formed.

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Chapter 1 - Types of Micro-organisms
By the end of this chapter you will be able to:

• Explain the classification of bacteria, fungi and viruses
• Explain how the structure of micro-organisms relates to their function
• Explain the growth and reproduction of micro-organisms
1.1 Bacteria
1.1.1 Bacterial Structure
Bacteria are simple one cell organisms which are found just about everywhere. Although
they can be a variety of shapes they all have the same basic features.
The cell wall is the strong outer layer which maintains the shape. It also acts as an initial
barrier to keep certain substances out of the cell. The cell wall acts like a sieve, keeping out
very big molecules, larger than about 1 nanometre. The cell wall is made up of
carbohydrate, cross linked by peptide chains.
The cell (or cytoplasmic) membrane is a very delicate structure which controls the entry and
exit of most substances. The cell membrane is able to selectively transport a range of
substances between the outside cell environment and the cell cytoplasm.
The cytoplasm is a viscous fluid containing vital cell components. The main items within this
are the ribosomes and nuclear materials. You may remember from the last module that
ribosomes are where the cell manufactures proteins.

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The nuclear material contains the genetic make-up of the cell in the form of DNA or RNA.
This nuclear material, the bacterial chromosome, is not surrounded by a nuclear membrane.
A plasmid is a small ring of very little DNA that bacteria can copy and pass onto other
bacterial cells. It is through plasmids that bacteria can pass on useful genes (such as
antibiotic resistance genes) and therefore improves their chances of survival.
Additional features of certain types of bacteria (but not all) are flagella, capsules, and the
ability to produce spores.
Flagella are thread like tails which start beneath the cytoplasmic membrane. The flagella
can move and this enables the cell to move about.
Capsules are produced by some bacteria. These are coatings which are bound to the cell
surface. Two of the functions of the coating are to protect the bacteria from drying out, and
also to prevent it from being ingested by white blood cells.
A Spore is a bacterial cell which has undergone a fundamental change and is in ‘hibernation’
mode. The spore is extremely resistant to unfavourable environments such as intense heat,
radiation and lack of nutrients. The spore can exist in a dormant state for years. When in a
favourable environment the spore may ‘germinate’ and become a normal bacterial cell.
In many medical situations things need to be sterilised to prevent infection. This means that
all micro-organisms have to be killed or removed including spores.
Why do you think bacterial spores make it difficult to sterilise things?
1.1.2 Classification of Bacteria
Bacteria can be classified in many different ways and are identified using a series of physical,
immunological or molecular characteristics.
Gram reaction
Gram-positive and Gram-negative bacteria are characterised based on the chemical and
physical properties of the bacterial cell wall. These bacteria respond differently to a gram
staining technique which allows them to be identified. These bacteria often respond
differently to different antibiotics.
Cell shape
Bacteria generally form one of three shapes. Coccus means sphere, bacillus is a rod shape
and spirillium is a spiral shape:

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A combination of cell shape and reaction to the Gram stain are frequently the first steps in
identifying bacteria. For example, Escherichia coli, a type of bacteria which can cause
urinary tract infections, is a Gram-negative rod. This means the Escherichia coli cells are rod
shaped and are not stained by the Gram stain. Staphylococcus aureus are gram positive
cocci, so they do stain with the Gram stain and are spherical in shape.
A range of other staining techniques are used to observe parts of the bacteria, such as
flagella, capsules and spores.
Atmospheric Preference
Aerobic organisms require oxygen to grow and live whereas anaerobic ones require an
atmosphere with very little or no oxygen. Organisms that grow in either atmosphere are
known as facultative anaerobes.
Helicobacter Pylori is a bacteria you will come across on this course as it can be the cause of
stomach ulcers. It is classed as a microaerophilic organism because it requires oxygen to
survive, but at lower levels than are present in the atmosphere. The stomach is therefore a
perfect environment for this microbe as it contains very little oxygen.
1.1.3 Growth and Reproduction of Bacteria – Binary Fission
Bacteria divide and grow through an asexual reproductive process called binary fission.
Since the genetic information (and the cell structure) of a bacteria is much simpler than that
of a eukaryotic cell, the process of binary fission is not as complicated as animal cell division.
1 2 3 4
5 6 7
8 9

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The single circular DNA chromosome of a bacterial cell first replicates then attaches each
copy to a different end of the cell membrane. The cell then begins to split (a stage called
cytokinesis) and as it does so the replicate and original chromosomes are pulled apart
resulting in two cells with identical genetic composition.
Due to this form of reproduction, all the bacteria in a colony would have been produced
from just one cell and therefore have the same genetics as each other. Therefore if a drug is
able to kill one type of bacteria then it will be effective against them all. However, if a
bacteria were to be resistant against a particular drug, then the whole colony would also
display resistance.
1.2 Fungi
Fungi are a very varied group of eukaryotic organisms. Remind yourself what eukaryotic
means from the previous section and complete the sentence:
Eukaryotic cells have ……………………. surrounding the genetic material, separating this from
the cytoplasm.
There is considerable variation in the structure, size, and complexity of various fungal
species. For example, they include simple one cell organisms (unicellular) like yeasts and
many multicellular organisms like moulds (US spelling - molds). Mushrooms are another
type of fungi, however we will not be covering them on this course.
1.2.1 Yeast
Yeasts are essentially similar in structure to bacteria, with the addition of a separate nucleus
surrounded by the nuclear membrane and eukaryotic organelles (you will learn about
organelles in the next unit). They frequently also have a vacuole filled with fluid.
There are many species of yeasts, the most well-known being Saccharomyces cerevisiae, the
common baker’s and brewer’s yeast. Much of what we know about cell and molecular
biology has come from studying this yeast.

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They reproduce asexually by a process of budding. In this process, a new cell forms at the
surface of the original cell, enlarges, and then breaks free to assume an independent
existence.
1.2.2 Moulds
Mould is a general term given to fungi growing as long, branching filaments of cells called
hyphae (singular hypha) that generates large numbers asexual spores for reproduction (or
sometimes sexual spores). These spores are often brightly colored, giving the tangles of
hyphae a distinctive appearance that may be useful in identifying the species. It is worth
noting that the spores produced by fungi are not as resistant to adverse conditions as
bacterial spores. Fungal spores are important in reproduction rather than protection.
Depending on the type of mould hyphae can function in absorption within the host cell,
transferring or exchanging nutrients, or to produce adhesive structures to aid growth.
Some common mould genera are Penicillium, Aspergillus, and Rhizopus.
Dimorphic fungi are those which have the ability to switch between the yeast and mould
forms depending on the environmental conditions they are growing in. Many fungal
pathogens exist in the body in the yeast form but revert to the mold form in the laboratory
when cultivated
1.3 Protozoa
These are simple one cell organisms. They live in a water environment such as in ponds,
ditches, soil and sea. Unlike bacterial cells they do have a nuclear membrane surrounding
genetic material, so they are eukaryotic organisms. They are the largest single cell (uni-
cellular) organisms. Some very large protozoa reach 2mm in length.

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Protozoa are able to move about by various means. Some have flagella; some have many
smaller tails called cilia all over the cell surface whilst others have a type of ‘feet’. They are
able to feed on organic matter by surrounding it and trapping it in within a vacuole where it
can be digested with enzymes.
Protozoa can reproduce by both asexual and sexual means, and some use a combination.
Most protozoa are harmless to man, but a few do cause disease. Malaria is caused by
protozoa, so is amoebic dysentery. In other countries, Giardia is one of the commonest
protozoal infections causing diarrhoea.
1.4 Viruses
These are the smallest micro-organisms. They are seen using an electron microscope which
magnifies in excess of 25,000 times. A normal light cannot give this much magnification.
Their actual size is about 10-300nm.
Viruses are very different from the other types of micro-organisms because they are entirely
parasitic. They can only grow when inside living cells. They cannot grow in any sort of
nutrient medium. To grow and reproduce they must take over the energy and protein
producing systems of a cell. They hi-jack the cell for their own use then once inside, make
hundreds of thousands of copies of themselves. Eventually the virus copies burst out of the
cell, killing it in the process.
As well as attacking animal cells, bacterial cells can also be targeted. Viruses that infect
bacterial cells are called bacteriophages or just phages.
Viruses are often specific to a particular type of cell. The influenza virus only reproduces in
the cells of the respiratory tract. Many viral infections in man are mild and may not produce
any symptoms. A few, like rabies are extremely serious. Some human viruses can cause
disease in other animals. Likewise we can catch some viruses from animals.
1.4.1 Virus Structure
Viruses are made up of a core of

genetic material inside a coat of
protein (called a capsid). The genetic
material of a virus can exist in either
DNA or RNA form. The virus (shown
right) is an influenza virus, and its
genetic information is coded as RNA.
The capsid protects the genetic
material from adverse environments
Some viruses also have a lipid
envelope covering the protein
capsid. This is usually derived from the host cell membrane and so can be used both as a

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tool for entering a host cell and also as a ‘camouflage cloak’ so that it is not recognised by
the host immune system.
The glycoprotein receptors are useful for initially binding to the outside of a host cell before
it enters. The influenza virus has two types of glycoproteins. One type binds the virus to a
host cell and the other type is thought to be predominantly involved in the release of newly
produced virus particles from the host cell.
1.4.2 Classification of Viruses
DNA Viruses are those that store their genetic information in the DNA form. This can either
be double-stranded or single-stranded. Examples include the adenovirus, herpes simplex
virus and the smallpox virus.
RNA Viruses are those which possess either single stranded or double stranded RNA as their
genome. These include rhinovirus, rubella virus, hepatitis C virus, influenza virus, measles
virus, mumps virus and the ebola virus.
Retroviruses are those which have an RNA genome, but also carry a special enzyme called
reverse transcriptase. Once in the host cell this enzyme converts the viral RNA into viral
DNA and the viral DNA is then cut and paste into the host’s DNA and viral proteins are
produced. The best example of this type of virus is the human immunodeficiency virus
(HIV).

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Take a Break!
I hope you first foray into the microscopic world was a
good one! Use this break to reflect on what you have learnt
so far.
Chapter 1 Summary
• Bacteria are prokaryotes and therefore differ from eukaryotes in that their genetic
information is not located within a nucleus. Instead it is a single ring of DNA. Other
features of bacteria are plasmids, flagella, capsules and their ability to form an extremely
resistant spore.
• Bacteria reproduce asexually through binary fission
• Bacteria can be classified due to their cell shape or the way in which they react to gram
staining.
• Fungi are eukaryotes and the microscopic forms include yeast and moulds.
• Yeast are simple and unicellular (have one cell) and reproduce asexually in a process
called budding
• Moulds grow forming long, branching filaments of cells called hyphae that generate
large numbers of asexual spores (different to bacterial spores) for reproduction.
• Protozoa are simple one celled eukaryotes which ‘eat’ organic matter by surrounding it
and enclosing the food within a vacuole. They can reproduce both asexually and
sexually.
• Viruses are not classed as either eukaryotes or prokaryotes. They consist of DNA or RNA
enclosed within a protein coat called a capsid. Others may have other features such as a
lipid membrane or glycoprotein receptors.
• Viruses can only grow inside other living cells, they hi-jack the cell and re-program it to
begin creating many new virus particles. After which the viruses burst out of the cell and
kill it.
• DNA viruses have DNA as their genetic material, RNA viruses have RNA as their genetic
material and retroviruses have an RNA genome but also a reverse transcriptase enzyme
which changes this genome from RNA to DNA once inside a host cell.

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Chapter 1 Quiz - Test Yourself

1. What is the definition of a prokaryotic cell?
a) The genetic information is surrounded by a nuclear membrane
b) The cell is not surrounded by a cell wall
c) The cell is surrounded by a cell wall
d) The genetic information is not surrounded by a nuclear membrane
2. Which of the following are prokaryotes?

a) Plant cells
b) Fungi
c) Bacteria
d) Protozoa
3. What type of micro-organism takes over a host cell and tricks it into producing many
copies of itself?
a) Bacteria
b) Fungus
c) Virus
4. A bacteria will have a...

a) Cell wall
c) Nucleus
c) Mitochondria
d) Cilia
5. A mould will have a....

a) Mesosome
b) Plasmid
c) Hypha
d) Capsid
6. A bacterial cell replicates by a process called what?

a) Multiple fission
b) Meiosis
c) Binary fission
7. A eukaryotic cell DOES NOT have...

a) Cell wall
b) Ribosomes
c) Capsid
d) Nucleus

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8. A virus can have...

a) Ribosomes
b) Mitochondria
c) Nucleus
d) Glycoproteins
9. What type of virus contains reverse transcriptase?

a) RNA virus
b) Retrovirus
c) DNA virus
10. What type of shape is a bacterium which has been categorised as a ‘bacillus’?
a) Sphere
b) Rod
c) Spiral
11. A bacterial spore is what?

a) Used for reproduction
b) Formed for bacteria to survive harsh environmental conditions
c) A toxin produced which causes disease
1.d; 2.c; 3.c; 4.a; 5.c; 6.c; 7.c; 8.d; 9.b; 10.b; 11.b
Chapter 1 Quiz answers

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Chapter 2 - Looking at Micro-organisms

• Describe the different types of microscopy used to view the microscopic world
Bacteria come in a range of shapes and sizes. Most are in the size range 0.8 – 4
micrometres. The most common shapes are spheres (coccoid) and cylinders (rod shaped),
but some have more exotic twists and spirals. When viewed under the microscope, many
bacterial cells are seen at the same time.
2.1 Light Microscope
Because of their very small size micro-organisms can only be seen

using a magnifying lens or microscope. A light microscope can
generally magnify to about 1200 times and the smallest things
you can see are of the order of 0.2µm.
Would you be able to see viruses with this type of microscope?
A light microscope can be used to

see living and non-living bacteria,
fungi and protozoa. The size, shape
and cell arrangement are visible. The image (right) is typical of
the view you might see when viewing bacteria through a light
microscope.
2.2 Electron Microscope
Electron microscopes are more powerful. These can only view non-living micro-organisms
as the specimen needs to be placed inside a vacuum to be able to view it (organisms cannot
survive in a vacuum). They cannot produce images in colour as electrons are used to
produce the image rather than light. A transmission electron microscope can be used to see
viruses and structures inside cells whereas a scanning electron microscope is used to
produce a 3D image, usually of the outside of an object. The image below shows a bird flu
virus taken using a scanning electron microscope:

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2.3 Other Forms of Microscopy
2.3.1 Phase Contrast
Phase contrast microscopy was described in 1934 by Dutch physicist Frits Zernike. It is a
technique used to produce very high contrast images without the need for staining and
fixing specimens (as with light and electron microscopy). Therefore transparent specimens
such as living cells can be viewed in their natural state to reveal dynamic biological
processes. It is this type of microscopy which allowed scientists to view cell replication
taking place.
This YouTube video shows a great example of phase contrast microscopy being used to view
cell division: http://bit.ly/phasecontrastmicroscopy
2.3.2 Dark Ground
Dark ground microscopy is a technique whereby the

unscattered light rays are excluded from the image to produce a
completely black background. This serves to illuminate the
object to great effect. The image to the right is an example of
this.
Dark ground microscopy can be used on live and unstained

samples, however the sample must be very strongly illuminated
to achieve a good contrast and this can sometimes damage the
sample.
2.3.3 Fluorescent
A fluorescent microscope is used to view samples that either naturally fluoresce or have
been treated so that they emit fluorescence. This is used routinely to determine gene
expression in cells whereby a gene is ‘tagged’ with fluorescent material. Fluorescence is
only emitted if the gene is being expressed by the cell.
However in addition to this, the structures of cells can also be ‘tagged’ to produce beautiful
imagery. Click this link to see a gallery of images which demonstrates the effects of
fluorescent microscopy:
http://bit.ly/fluorescenceimages

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Take a Break!
Scientists have learnt so much about the microbe world
through the use of different types of microscopy. I hope
you enjoyed learning about these.
Chapter 2 Summary
• A light microscope uses light to create the magnification and is used to view living and
non-living body tissue and bacteria in colour. However the magnification is limited to
about 2000x and viruses are too small to be viewed using this method.
• An electron microscope is very powerful and uses electrons to create the magnification.
This allows a magnification of up to 2,000,000x. However this means that only non-living
specimens can be viewed and colour cannot be seen.
• A transmission electron microscope is used to see structures within cells, whereas a
scanning electron microscope is used to produce a 3D image of the outside of a
specimen
• Phase contrast produces high contrast images without the need for staining so dynamic,
transparent images are produced.
• Dark ground microscopy is produced by powerfully illuminating the specimen and then
excluding any unscattered light rays. This produced attractive images however the
strong illumination may damage a sample
• Fluorescent microscopy is used to view samples in which certain proteins or structures
have either been ‘tagged’ with fluorescence, or emit their own fluorescence. This is
typically used to investigate the expression or role of a single structure or protein in a
cell.

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1. Bacteria are the smallest micro-organisms. True or false?
a) True
b) False
2. Which type of microscopy has the highest magnification?

a) Electron
b) Phase contrast
c) Fluorescent
3. Which type of microscopy produces high contrast images of specimens of which it is

possible to view biological processes taking place?
a) Electron
b) Fluorescent
c) Phase contrast
4. Which type of microscopy produces 3D images?
a) Light microscopy
b) Scanning electron microscopy
c) Transmission electron microscopy
5. A disadvantage of electron microscopy is....

a) You cannot view biological processes taking place
b) The magnification is limited
c) You cannot view the insides of a cell
1.b; 2.a; 3.c; 4.b; 5.a


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Chapter 3 - Growth of Micro-organisms

• Explain the factors that affect the growth of micro-organisms.
• Describe a bacterial growth curve.
• Explain the different methods of bacterial counting.
• Explain the uses of different growth media
Growth of bacteria is measured in terms of population counts. When a bacterial cell reaches
a certain size, it will divide into two daughter cells. This process is repeated many times so a
colony of cells is formed. So long as favourable growth conditions are maintained, growth
continues, and more and more cells are produced. This increase in numbers can occur very
quickly, the time between one cell division and the next may be as little as 10 minutes.
If you started with one bacterial cell and cell division occurred every 10 minutes,
how many cells would there be after half an hour?
Start 1 cell
10 minutes 2 cells
20 minutes 4 cells
30 minutes 8 cells
And after an hour and a half there would be 512!
3.1 Bacterial Counting
Counting bacteria in a sample is required for many experiments and investigations. For
example, analysing water samples, food samples or looking at how effective a disinfectant is
at killing bacteria. We will look at two different ways of counting bacteria.

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3.1.1 Direct Microscopic Counts
These are possible using a special counting slide known as a haemocytometer (or counting
chamber). It is called a haemocytometer as it was originally intended to count red blood
cells, however it is now used to count many cell types. The haemocytometer looks like this:
Each large red square represents 1mm2. The large squares are then subdivided into smaller
squares (yellow) of size 1/16mm2. The smallest squares (blue) are located in the middle of
the slide and are of the size 1/256mm2.
Large red square = 1.0 mm2

Medium yellow square = 0.0625 mm2
Small blue square = 0.0039 mm2
In this method a sample in placed on the haemocytometer slide and then viewed under a
light microscope. The square that is used to count the cells usually depends on how densely
populated the sample is with bacteria.
Next, the number of bacterial cells in one square is counted and the number is multiplied up
to find the number of bacterial cells per ml (millilitre).
The depth of the haemocytometer is known to be 0.1mm and therefore we can work out
that each mm2 is the equivalent to 0.0001ml (millilitres).
Therefore the volume represented by the small blue square is only 0.00000039ml (this is
worked out by the calculation 0.0001 ÷ 256).

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So to work out the number of cells per ml scientists would count the number of cells in the
small blue square and multiply by 2564102.56 (this is because 1 ÷ 0.00000039 = 2564102.56)
Example:
Don’t worry about the maths – just understand that you would count the number of
cells in the small squares and then multiply up accordingly to get the number of cells
per ml
A student wanted to find out the number of bacterial cells in a sample of pond water. She
places a sample on the haemocytometer and views it under a microscope. She counts 76
bacterial cells within one small 1/256mm2 square. How many cells are present per ml?
Calculation:
Number of cells per ml = 76 x 2564102.54

= 194871795 bacterial cells
Therefore approximately 195 million!
3.1.2 Indirect Viable Cell Counts
These are also called plate counts, and it involves plating out a sample on a nutrient agar
surface.
This picture shows a plate

with a number of colonies
growing on it. Each white
dot is 1 colony forming unit
(1 cfu)
If the nutrients in the media are suitable then each bacterial cell will grow and form a colony
of cells (these are a group of bacterial cells which have all grown from a single bacterium
and are therefore genetically identical). Each colony that can be counted is termed a colony
forming unit (cfu).

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Although this is not an exact count of the number of bacteria present, the number of cfus
will be related to the total number of viable bacteria in the original sample. Therefore plates
can be compared with each other for investigations. This method will only count the number
of bacteria that are living (hence the name viable) as only living bacteria will grow to form
part of a colony. This is unlike direct microscopic counts where it is impossible to tell
whether you are counting a living or dead bacterial cell.
3.2 Growth Curves
A growth curve is intended to show the relative number of bacteria living in a population
over time.
Plotting a graph of bacterial numbers or 'population count' against time is difficult because
the numbers soon disappear off the scale, or the scale is so great that small numbers are
impossible to plot. Because of this, graphs are plotted using something called a logarithm
graph. This produces the characteristic graph shown in the graph below. Don’t worry about
the term ‘log’ – it’s not easy to explain let alone understand!
Part A – Lag Phase

The first part of the graph (A) is the lag phase. When the bacteria is first put in a cell free
(sterile) medium it takes some time to get used to the environment. The cell absorbs
nutrients, makes enzymes and prepares for reproduction. Depending on the cell and the
environment this lag time may vary from minutes to hours.

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Part B – Log Phase

The second part is the log phase (B) when growth occurs. The population doubles at each
cell division. This log growth phase continues whilst ideal growth conditions are
maintained. This is not usually for very long because the nutrient supply is used up and
waste products build up. The growth phase can only be maintained if nutrients are
constantly added to the medium and waste products removed.
Part C – Stationary Phase

The third part of the graph (C) is the stationary phase. Here, the build-up of waste products
and lack of nutrients result in a slowdown in the rate of cell division. In the stationary
phase, the number of bacteria dividing equals the number dying so the total number of live
organisms remains the same.
Part D – Death Phase

In the death or decline phase (D) there is a lack of nutrients, further build-up of waste
products and the cells die. In the case of bacteria which form spores, this is the time when
spores will be formed so the bacteria can survive until there is a change in the
environmental conditions.
3.3 Environmental factors and growth
The growth of micro-organisms is affected by various environmental factors including

nutrition, pH, temperature, osmotic pressure, atmospheric pressure and oxygen.
3.3.1 Nutrition
All living organisms have to have the six chemical elements: carbon, hydrogen, oxygen,
nitrogen, phosphorus and sulphur. Most also need magnesium, iron, sodium, potassium,
chlorine, calcium and iodine. These are the elements from which the essential molecules -
carbohydrates, fats, proteins, DNA and RNA - are made. Organisms need a source of energy
to build these elements into the compounds they need. Any compound that the organism
cannot make for itself is called an essential nutrient. Different micro-organisms have
different essential nutrients.
Large molecules must be broken down before they can be taken into the cell, so many micro
-organisms release enzymes into the environment to do this. Inside the cell, nutrients are
used as building blocks to make molecules the organism needs by the process called
metabolism. Metabolism requires an energy source, and this is commonly a sugar, or, for
plants, sunlight. Metabolism is the term for the way cells chemically change food so that it
can be used to keep the organism alive.

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One part is called catabolism when the body actually uses food for energy. The other
process is called anabolism when the body uses the food to build or mend cells.
3.3.2 Temperature
There is an optimum temperature at which each organism grows best, but this is very
variable. Similarly there are minimum temperatures below which there is no growth and
maximum temperatures above which there is no growth.
Organisms that we associate with disease in man tend to grow best at body temperature
(37°C). But there are organisms with optimum growth temperatures close to freezing and
those that grow best at temperatures above 45°C.
The optimum temperature is the one at which the bacteria’s enzyme function best at. If the
temperature is below the optimum then bacterial reactions will occur more slowly and
therefore growth will be slower. If the temperature is raised above the optimum then it will
begin to harm the bacterial enzymes and affect its structure so that bacterial reactions
necessary for growth cannot occur. Enzymes are proteins after all, so think back to Module 4
for more detail of how temperature affects proteins.
Micro-organisms that can survive freezing conditions are called psychroduric and those that
can survive boiling are called thermoduric.
3.3.3 pH
pH is an important factor in microbial growth. Most prefer a neutral growth medium.

However, there are microbes that thrive in either acid (acidophilic) or alkaline (alkalophilic)
conditions.
Complete the following to remind yourself about pH.
Most microbes prefer a neutral medium with a pH value of about --------------
Microbes that thrive in a medium of pH 3 are called ------------------.
Microbes that thrive in a medium of pH 9 are called---------------------
Think about the pH in the gastrointestinal tract.
Which type of organism would you expect to find in the stomach? (The contents of the
stomach are acidic).
3.3.4 Oxygen
The word respiration can be used to mean breathing - taking air in and out of the lungs. But
inside the organism or at the cellular level respiration is the chemical reaction from which an
organism derives energy.

22
Most micro-organisms are aerobes. This means that they need oxygen for respiration. They
grow best in an atmosphere containing oxygen. However there are organisms (anaerobes)
that do not need oxygen for respiration. Anaerobes actually die if exposed to oxygen.
Microbes that can adapt to atmospheres with or without oxygen are called facultative
organisms.
The organism Clostridium perfringens which causes gas gangrene is an obligate (it must be)
anaerobe. For this reason treatment has included putting the patient in a chamber with
high oxygen pressure: exposure to oxygen kills the infecting organism.
3.3.5 Water and osmotic pressure
All micro-organisms need water for growth. Nevertheless none can grow in pure water
where there is no food present. Most bacteria grow best in dilute solutions; yeasts can
survive stronger solutions and fungi the strongest.
The osmotic pressure of the solution is also very important.
Bacterial cells are subject to the same osmotic forces as any cells in the body.
When the bacterial cell is placed in a hypertonic solution (a solution which has a high solute
concentration) it becomes dehydrated as water is drawn out of the cell into the surrounding
medium. This causes the cell membrane and cytoplasm to shrink away from the cell wall.
This process is called plasmolysis and it inhibits cell growth.
Plasmoptysis occurs when a bacterial cell is placed in a hypotonic solution (a solution which
has a very low solute concentration). Water enters the cell, moving from the weaker
external medium to the more concentrated cell cytoplasm. The cell wall prevents the cell
swelling so the internal pressure builds up. This pressurised state is called plasmoptysis.
3.3.6 Trace Nutrients
Trace nutrients are metal ions required by certain cells in such small amounts that it is
difficult to detect (measure) them, and it is not necessary to add them to culture media as
nutrients. Trace elements are required in such small amounts that they are present as
"contaminants" of the water or other media components. As metal ions, the trace elements
usually act as cofactors for essential enzymatic reactions in the cell. One organism's trace
element may be another's required element and vice-versa, but the usual ions that qualify
as trace elements in bacterial nutrition are manganese, cobalt, zinc, copper, and
molybdenum.
3.4 Laboratory Media
Bacteria are cultured in the lab by growing it in some sort of laboratory media – this is a
substance which contains all the nutrients needed for growth. Culturing bacteria from a
sample in the body can indicate that it is involved in a disease process and it is also the first

23
step in studying its morphology. As discussed earlier, plating out a sample on nutrient agar
is also a way to estimate bacterial cell growth (by counting their colony forming units)
Laboratory media can come in various forms and types.
3.4.1. Different Media Consistencies
Liquid Culture Media

This type of media is used for growing bacteria in test tubes, bottles and flasks. It is
sometimes referred to as a ‘broth’ (e.g. nutrient broth). The bacteria grow throughout the
liquid and this type of media tends to be used when large numbers of bacteria need to be
grown. It also helps to dilute any inhibiting factors to bacterial growth, however the
properties of the bacteria cannot be viewed in this type and different types of bacteria
cannot be detected.
Solid and Semi-solid Culture Media

A solid culture media is produced from a liquid medium by the addition of a solidifying agent
called agar. The media is then poured into a Petri dish where it is left to solidify. Whether
the media is solid or semi-solid depends on the amount of agar added. A solid culture media
is made by adding 1-3% agar, whilst reducing the amount of agar to 0.2-0.5% renders it
semi-solid.
The advantage of using this type of culture is the ability to identify and characterise the
different colonies which grow. Scientists are also able to pick one of these colonies and then
grow a pure culture. It is also possible to estimate the number of bacteria in a sample by
counting the colony forming units (cfus).
3.4.2 Different Media Components
Selective Media
Selective media is prepared by adding a component that will inhibit the growth of
unwanted, contaminating bacteria but will not affect the bacteria you are trying to grow.
Different approaches to make media selective include the addition of antibiotics, dyes,
chemicals, alteration of the pH, or a combination of these.
Differential Media
This type of media incorporates dyes and metabolic substrates which are only used by the
bacteria which you want to identify. In doing this, the bacteria will utilise the coloured
component and then will form differently coloured colonies that are instantly recognisable.
Enriched Media
Enriched media is used to grow nutritionally demanding bacteria as it contains the addition
of extra nutrients such as blood, serum and egg yolk etc.

24
Take a Break!
Time again for another break from learning. You should
now understand more the factors which govern microbe
growth and have some knowledge on the tools and
techniques scientists employ to grow and count microbes
in the laboratory.
Chapter 3 Summary
• Microbes will grow in any conditions that are favourable to them. In order to grow they
require nutrients for energy and growth, water, trace nutrients and a temperature and
pH optimal for them. Different microbes may have a different optimum temperature
and pH
• Aerobic microbes require oxygen for respiration, whereas anaerobes are poisoned by
oxygen.
• Osmotic pressure is also a factor for growth. A hypertonic solution (a solution which has
a high solute concentration) causes dehydration as water moves out of the cell; and a
hypotonic solution causes water to move into the cell leading to a highly pressurised
state.
• The size of a bacterial population can be found by taking a sample and using a bacterial
counting technique. A direct microscopic count method involves placing the sample
onto a haemocytometer, viewing the sample under a light microscope, then counting
the number of cells within a small area, before multiplying up to achieve a value per
millilitre. An indirect viable cell count involves growing a sample on an agar nutrient
plate then counting the number of colony forming units (cfu) which form.
• The growth of a bacterial population over time can be plotted on a growth curve. This
curve will show the different population phases – lag phase, log phase, stationary phase
and death phase.
• Scientists can grow microbes in the laboratory using different types of growth media
depending on the type of microbe they are growing and what they are investigating.
Nutrient media can be liquids, solids or semi-solids, and selective, differential or
enriched.

25

1. What is the log phase of a bacterial growth curve?
a) The phase where the bacterial population neither increases nor decreases
b) The phase where the bacterial population increases
c) The phase where the bacterial population decreases
2. What is selective nutrient media used for?

a) To inhibit the growth of unwanted microbes so that only the desired bacteria will grow
b) To colour the desired bacteria so that it is more selectable
c) To provide your desired bacteria with the extra nutrients it needs for growth
3. An anaerobe is a type of microbe which....

a) Is poisoned by oxygen
b) Requires oxygen to survive
c) Requires oxygen to survive but only in very small amounts
4. All microbes will grow best in what conditions?

a) High temperature and pH
b) Low temperature and pH
c) Optimum temperature and pH
5. Which is the most inaccurate method of counting the number of bacterial cells in a
sample?
a) Microscopic count using a haemocytometer
b) Viable cell count
c) They are both as accurate as each other
6. What does one white spot on this nutrient agar plate (right)
represent?
a) One virus colony
b) One bacterial cell
c) One colony forming unit
7. A hypertonic solution will cause what?

a) Dehydration
b) Plasmoptysis
c) Replication
1.b; 2.a; 3.a; 4.c; 5.b; 6.c; 7.a


26
Chapter 4 - Microbial Control of the Environment

• Describe methods used to control and monitor the microbial content of the
environment, including environment and personal hygiene.
• Describe the different groups of disinfectants and explain how disinfectant activity
may be tested in the laboratory.
4.1 Environmental Control
Microbes are present in almost every environment on earth! In buildings, such as your
pharmacy, they are usually present in low concentrations on surfaces or as floating dust
particles.
4.1.1 Airborne Control
The numbers of airborne microbes can be controlled through fitting an air filtration system.
The filter pores will be small enough to trap the microbes but large enough to allow the free
flow of air. Improving air circulation will also help as airflow will be more evenly distributed
and therefore the effectiveness of air cleaning is maximised.
4.1.2 Personal Hygiene
Harmful bacteria live in and on our bodies, especially on and around our faces and hands,
and on our clothing. As they are usually present in small numbers they do not make us ill. If
these bacteria are transferred from our bodies or clothes onto food, and allowed to
multiply, the food can become unsafe to eat. In hospital there are lots of people who are
coughing and sneezing, suffering with infected weeping and draining wounds. Some of the
individuals are immunocompromised (meaning that the ability of their immune systems to
fight off infections has been weakened), some are elderly and frail. There is movement all
day long. Nurses go from room to room, all sorts of equipment are wheeled in and out,
food is delivered on trays, trollies go from ward to ward, physicians make rounds, patients
touch rails, toilets, handles… It’s really not surprising that some people catch something that
they did not have when they went in.
Microbial control of our body surface centres around regular handwashing and the use of
antiseptics.
4.1.3 Equipment and Work Surfaces
Bacteria are able to live and multiply in any cracks and crevices in equipment. Surfaces such
as bench tops may have bacteria on them from contact with people, dirty equipment or
other things such as cartons that have been stored on the floor. If the bench tops are not
properly cleaned, anything placed on them will be contaminated by the bacteria.

27
Cleaning is the process that removes contaminants including dust, solid, large numbers of
micro-organisms and organic matter such as blood or vomit. It is an essential prerequisite to
disinfection and sterilisation. It also removes the micro-nutrients on which micro-
organisms might subsequently thrive.
Hospitals should have their own disinfectant policies and guidelines. An example hospital
policy for management of diarrhoea spillages is given in the flow chart on the next page.
Prior to such guidelines, chemical disinfectants tended to be used haphazardly with little
consideration for appropriate strengths and purpose. Most jobs can be done with one
standard solution.
Flow Chart Showing the Policy for the Management of Diarrhoea Spillages
Body fluid/diarrhoea spillage

Nurse puts on sterile gloves and apron
Remove spillage using disposable paper, not mop
Discard into yellow plastic waste sack

Clean area with detergent and hypochlorite (560ppm) and paper roll
Do not use mop
Discard into plastic waste sack
Remove apron and gloves

Discard into yellow plastic sack
Wash hands
If you work in a hospital, the pharmacy or microbiology departments will probably have
copies of that hospital’s disinfection policy - Look through to find out:
a) What chemical disinfectants are used?
b) What procedures are used for bedpans, stethoscopes and a range of other items?
4.1.4 Healthcare Associated Infections (HCAI)
Although better infection control policies have resulted in the rates of many hospital
acquired infections falling quite significantly over the past few years, some patients still
contract infections during stays in hospital. Risk factors for infection include implants,
diabetes, smoking, poor nutrition, prolonged pre-operative hospitalisation and prolonged
surgery.

28
Though cleanliness contributes to infection control HCAI can be inevitable, particularly in

patients who are old, frail or have undergone complex medical procedures. The most
common cause of HCAI is the bacterium Staphylococcus aureus. Many of us have this
bacterium in our noses or on our skins. For approximately 50 years Staphylococcus aureus
infections were treatable with Penicillins, for example Cloxacillin and Flucloxacillin.
However, through evolution, strains of Staphylococcus aureus have become resistant to such
drugs. Currently, the most publicised is Methicillin Resistant Staphylococcus Aureus, or as it
is more commonly known, MRSA.
Media coverage suggests that MRSA is more prevalent than other forms of Staphylococcus
aureus infections but there is currently insufficient data available to confirm whether this is
true. Patients who develop infections caused by MRSA are either colonised with the
bacterium at the time of their admission to hospital, acquire it through surgery or through
cross infection post-surgery. Risk factors for MRSA include previous hospital admissions,
pressure sores, underlying disease, implants, intravascular lines and antibiotic
administration. Certain strains of MRSA, are easy to spread between patients and can
colonise in debilitated patients. MRSA does not seem to be a problem in healthy patients.
Trauma patients and elderly patients are seen to be most at risk.
Thankfully, cases of MRSA are now falling significantly, after a period during which it seemed
to be on the increase. There is now much better reporting and more recognition of the
infection. However, other types of infections such as Clostridium Difficile are now causing
problems and there is a worrying increase in the numbers of infections, both bacterial and
viral, that are becoming resistant to antimicrobials, meaning that some infections are
becoming much more difficult to treat.
There is also difficulty in containing the spread as the source of infection can be from the air,
visitors, surgical instruments, hospital environment, the hands and people working within
the hospital environment. Within hospitals there are limited isolation facilities to cope with
the number of cases with layouts and designs of hospital buildings not lending themselves to
isolation and in the past, there was probably a lack of importance and resources placed on
infection control measures and general hygiene.
Which actions/products would you recommend to reduce the presence of problem bacteria
in:
a) the home?
b) the workplace?

29
For the last few years the Health Protection Agency (now part of Public Health England) has
been publishing annual rates of MRSA and other HCAIs and advising NHS Trusts to display
their own rates and trends in public areas and to include them in their published annual
reports.
You can find the most recent reports on HCAIs from the following link on Public Health
England’s website: https://hcaidcs.phe.org.uk/
4.2 Sampling Techniques
For an indication on how well the microbe growth is being controlled there are various
sampling techniques in which a sample is taken from the air or a surface
4.2.1 Settle plates
This involves leaving a standard Petri dish with nutrient agar out in the open (with the lid
off) for a given period of time. The plates are then covered up and incubated to allow visible
colonies to grow and be counted. This is a passive technique for monitoring the microbe
population in the air.
4.2.2 Air Samplers
An air sampler, as the name suggests, is also used to monitor the microbe population in the
air, but it is a more active technique. An air sampler draws a known volume of air, using a
pump or fan, through a perforated plate and onto a collecting surface (normally an adhesive
medium such as agar). The perforated plate means that the velocity and spread of air onto
the collecting surface is controlled and always the same. When the correct volume of air has
passed through the sampling head, the agar plate can be removed and incubated directly.
4.2.3 Swabs
A swab can be used to sample both body surfaces and hard surfaces. The swab should be
moistened with a solution that neutralises any detergents and sanitisers and also preserves
the integrity of the sample i.e. the bacterial numbers should be stable. The swab is then
used to transfer the sample onto an appropriate nutrient agar plate.
4.2.4 Contact Plates
Contact plates are a plastic dish filled with nutrient agar to give a convex surface. The
contact plate is then pressed directly onto the surface that is being sampled, before being
covered and incubated. They are mostly used for surface sampling but they can also be
placed into some air samplers.

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4.3 Disinfectants and Antiseptics
Disinfection is a process which destroys micro-organisms or reduces numbers to the point at

which they are unlikely to cause diseases. Disinfection does not usually affect bacterial
spores. This contrasts with sterilisation where all organisms are destroyed.
An antiseptic often contains the same antimicrobial agent as a disinfectant but it is usually
present in a lower concentration and therefore is safe to use on the skin. Some antiseptics
are capable of destroying micro-organisms, but most work by inhibiting or preventing their
growth.
Some agents are active against a wide spectrum – others are not.
A disinfectant is a chemical used to destroy or inhibit microbial growth on non-living

materials e.g. floors, work surfaces, equipment and so forth. Many household products
such as cleaning materials and hand sanitisers contain disinfectants. Disinfectants are also
used to clean items such as contact lenses and babies’ bottles
In practice, disinfectants are used to reduce the number of micro-organisms to acceptable,

non-harmful levels. Control of micro-organisms is vital in hospitals and many areas of
industry such as pharmaceuticals and food. Knowledge of the anti-microbial action of the
various agents available is important in the selection of an appropriate agent for a particular
situation. In hospitals, successful disinfection also depends upon an understanding of how
infectious diseases are transmitted. For example, where diseases are spread by skin
contact, the need to disinfect the hands of nurses and others who touch the patient is clear.
Problems with chemical disinfectants include:
• Inactivation by other chemicals such as soap and detergents

• Ineffective concentrations
• Limited activity against some types of organisms
• Inactivation by organic material
• Short shelf life once opened or made up
• Prolonged contact time may be needed to be effective
• Washing required prior to use
• Made up incorrectly or inactivated in some way, most disinfectant solutions can
actually be colonised by bacteria- over dilution will render them inactive
• Need to be used at the correct pH
• Need to be used at the correct temperature
For those of you who have a keen interest in this topic, perhaps because you are working in
a manufacturing environment, you might like to read more about choosing the right
disinfectant:
http://bit.ly/choosingdisinfectants

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4.3.1 Antiseptics and disinfectants in medicine and pharmacy
The + indicates some activity against each type of organism.
Agent Example Activity Fungi Bacteria Spores Viruses
Aldehydes FORMALDEHYDE Active against a + + + +

wide range of
micro-organisms
Irritant and
hazardous
Halogen releasing CHLOROS® Household + + limited +
e.g. hypochlorite, bleaches of
iodine choice for action
against HIV and
Hepatitis B
Alcohols Ethanol Limited use but some + none some
active against
suspensions
containing
Mycobacterium
tuberculosis
Biguanides Chlorhexidine Broad spectrum + + none limited
Quaternary Benzalkonium varying + sporostatic limited

Ammonium chloride
Compounds (QACs)
Peroxygen Hydrogen peroxide + + +
Phenols STERICOL® + + none limited
4.3.2 Alcohols (e.g. Ethanol)
Alcohols exhibit rapid broad-spectrum antimicrobial activity against bacteria, viruses, and
fungi but do not kill bacterial spores. They are widely used for both hard-surface
disinfection and skin antisepsis. Lower concentrations may also be used as preservatives.
Little is known about the specific mode of action of alcohols, but based on its increased
effectiveness in the presence of water it is generally believed that they cause membrane
damage and rapid denaturation of proteins. This subsequently disrupts metabolism and
causes the bacterial cell to burst.
4.3.3 Aldehydes (e.g. Formaldehyde)
Formaldehyde is a reactive chemical that interacts with protein, DNA, and RNA. It has been
proposed that formaldehyde acts as a mutagenic agent because it reacts extensively with
nucleic acid. It has shown the ability to form protein-DNA cross-links thereby inhibiting the
DNA synthesis of bacteria. It is able to kill bacterial spores due to its ability to penetrate into
their interior.

32
4.3.4 Biguanides (e.g. Chlorhexidine)
Chlorhexidine is probably the most widely used antimicrobial in antiseptic products, in

particular in handwashing and oral products but also as a disinfectant and preservative. This
is due in particular to its broad-spectrum, effectiveness and low irritation.
It works by damaging the cell membrane causing partial leakage of the intracellular
constituents. High concentrations also cause the constituents of the cell to coagulate
together, congealing the cytoplasm and thus inactivating cell processes.
4.3.5 Halogen Releasing Agents (e.g. CHLOROS®)
This group includes both chlorine releasing agents, used for hard surface disinfection
(household bleach) and disinfecting spillages of blood containing HIV, and iodine which has
been used as an antiseptic for over 150 years.
The mechanism of action for these is not entirely known. The chlorine releasing agents are
highly oxidizing and this can destroy cellular proteins. It is also possible that this property
induces mutations in bacterial DNA. Iodine is able to rapidly penetrate into microbes and
has been shown to attack key groups of proteins, nucleotides and fatty acids, which
ultimately results in cell death.
4.3.6 Peroxygens (e.g. hydrogen peroxide)
Hydrogen peroxide is a widely used biocide for disinfection, sterilisation and antisepsis and
has broad spectrum activity against viruses, bacteria, yeasts and spores. It acts as an oxidant
and attacks essential cell components including lipids, proteins and DNA.
4.3.7 Phenols (e.g. STERICOL®)
Phenols kill bacteria by causing membrane damage at the point of cell division, therefore
inducing cell leakage. It is probably this ability to damage the membrane which provides
their anti-fungal properties too. Phenols have an effect against some viruses too,
particularly those which have lipid envelopes. Phenols are not effective against non-
enveloped viruses and spores
4.3.8 QACs (e.g. benzalkonium chloride)
Quaternary ammonium compounds (QACs) have been used for a variety of clinical purposes
such as the pre-operative disinfection of unbroken skin. In addition to having anti-microbial
properties, QACs are also excellent for hard-surface cleaning and removing odours.
QACs are membrane-active agents. They penetrate into the cell wall, react with the
cytoplasmic membrane (lipid or protein), cause cell leakage, then go on to degrade proteins
and nucleic acids. There is thus a loss of structural organisation and integrity of the
cytoplasmic membrane in bacteria, together with other damaging effects to the bacterial
cell.

33
4.4 Sterilisation
The complete destruction of all living microbes including cells, spores and viruses is called
sterilisation. The main methods for sterilisation are heat, autoclaving (steam plus heat),
chemicals, filtration and radiation. A sterilant is a chemical agent used in sterilisation.
This can be achieved by steam, a combination of steam and formaldehyde, hot air, ethylene
oxide or irradiation.
Autoclaving is the most common method. It uses steam under pressure.
Formaldehyde is an effective sterilant, but has to be used with caution because it causes
irritation to the eyes, respiratory tract and skin and its use has been linked to nasal and lung
cancer. It can be absorbed by some materials and then slowly released with potentially
damaging effects.
Ethylene oxide is a colourless gas which is toxic if inhaled. It is effective against all organisms
and does not damage equipment. It is particularly useful for sterilising products and
equipment that would be damaged by high temperature sterilisation. A downside is that it
is relatively expensive.
Sterilisation by irradiation is generally reserved for industrial-scale sterilisation. Gamma

radiation is often used to sterilise disposable medical equipment such as syringes and
needles but can cause serious deterioration of certain materials. Electron beam processing
uses higher doses of radiation but exposure time is less, so there is less potential for
degradation. High energy X-rays are used to sterilise larger pieces of equipment.
4.5 Cross-Contamination
Cross-contamination is when bacteria or other microbes are spread between food, surfaces
or equipment. It's most likely to happen at home when:
• raw food touches (or drips onto) other food
• raw food touches (or drips onto) equipment or surfaces
• people touch raw food with their hands
If you cut raw meat on a chopping board, bacteria will spread from the meat to the board
and knife. If you then use the same board and knife (without washing them thoroughly) to
chop a cucumber, the bacteria will spread from the board and knife to the cucumber.
Hands can also spread bacteria. If you touch raw food and don’t wash your hands
thoroughly you can spread bacteria to other things you touch.

34
Take a Break!
Don’t worry if you feel like that was a lot to take in. Take a
quick break, read the summary below and go over the main
points again if you need to.
Chapter 4 Summary
• Microbes are present in almost every nook and cranny on earth! They appear in the air
and on our body and work surfaces.
• Their numbers in the air can be controlled in the air through implementing an air
filtration system, and improving air circulation. They are sampled by using settle plates
or air samplers
• The growth of microbes on non-living surfaces such as work benches and floors is
controlled be regular cleaning, and disinfecting or sterilising with the appropriate
chemical agent. Your hospital or pharmacy will have their own disinfection policies
which I urge you to have a look at.
• Disinfection is a process which destroys or reduces the numbers of micro-organisms, but
does not usually affect bacterial spores. This contrasts with sterilisation where all
organisms are destroyed.
• Antiseptics are used to control microbial growth on living tissue. They usually contain
the same antimicrobial agents as disinfectants but in lower concentrations.
• Microbial growth on living or non-living surfaces is sampled using swabs or contact
plates.
• Disinfectants include the alcohols, aldehydes, biguanides, halogen-releasing agents,
peroxygens, phenols and QACs. Each of these have their own individual spectrum of
activity and mode of action.
• Sterilisation involves the complete destruction of all microbes including bacterial spores.
Autoclaving (treatment with steam under pressure) is a widely used method of sterilising
equipment (laboratory equipment or surgical instruments). Irradiation, formaldehyde,
filtration and ethylene oxide can also be used.

35

1. What type of medication is used to kill bacterial infections?
a) Fungicides
b) Disinfectants
c) Antibiotics
d) Fungibiotics
2. Autoclaving and radiation are types of what?

a) Preservatives
b) Sterilisation
c) Disinfection
d) Growth techniques
3. Sterilisation is....
a) The complete destruction of all living microbes including spores and viruses
b) A chemical used to destroy or inhibit microbial growth on non-living materials
c) The complete destruction of all living microbes except spores
d) None of the above
4. A settle plate is used to collect microbes from where?

a) The air
b) Body surfaces
c) Floor
5. Which of these is a method used to collect a sample of microbes from both living and
non-living surfaces?
a) Air sampler
b) Swab
c) Settle plate
6. What is an antiseptic?
a) A low concentration disinfectant used for personal hygiene
b) A high concentration disinfectant used for sterilisation
c) A disinfectant which can only be used on non-living surfaces
7. Formaldehyde can kill all types of micro-organisms

a) True
b) False

36
8. Ethanol can kill all types of micro-organisms

a) True
b) False
9. Chlorhexidine is a broad spectrum disinfectant

a) True
b) False
10. Which disinfectant works by forming DNA-protein cross-links and inhibiting DNA
synthesis?
a) Chlorhexidine
b) Ethanol
c) Formaldehyde
1.c; 2.b; 3.a; 4.a; 5.b; 6.a; 7.a; 8.b; 9.a; 10.c

37
Chapter 5 - Infectious Diseases

• Describe diseases caused by pathogenic micro-organisms
• Explain the role of micro-organisms in the transmission of disease and infections
5.1 Cause and Transmission of Infection
The first stage in infection is for the pathogen to enter the body. So how do the micro-
organisms get in? Micro-organisms cannot normally get through the skin. However if the
skin is damaged by cuts or burns microbes may enter. If the skin is penetrated by insect or
animal bites, or by injection of contaminated material, microbes may enter. Occasionally
infection of the skin itself may occur e.g. ringworm is caused by a fungus on the skin.
The easy way in for micro-organisms is through the body openings, i.e. the nose and mouth,
anus, urogenital tract, ears and eyes. These sites do have their own protection mechanisms
but some micro-organisms still get through.
There are six main ways that infections are transmitted:
5.1.1 Directly by person to person skin contact
For a disease to be transmitted by direct contact

the causative organism is carried on the skin.
Diseases transferred in this way include colds,
influenza, tuberculosis, polio, and diphtheria. This
type of transfer is very common in hospitals.
5.1.2 Directly by intimate person to person mucus contact
The intimate transfer of infections from mucus to mucus is

also called the venereal route. Sexually transmitted
infections (STIs) like syphilis and gonorrhoea are only
transmitted in this way. Other diseases like HIV and
hepatitis can be transferred by this and other routes.
5.1.3 Direct blood contamination by non-sterile syringes and needles, intravenous

infusions or kidney dialysis
Many diseases can be transferred by non-sterile instruments e.g. HIV and

hepatitis

38
5.1.4 Indirectly by dust or droplets in the air
Contagious airborne diseases are often due to pathogens in

water droplets in the air. Diseases carried by water and dust
in the air include colds, influenza, measles, mumps, and
rubella.
5.1.5 Indirectly by contamination of food or water
Contamination of food and water is common. Sewage may inadvertently

contaminate supplies of drinking water; food may be carelessly handled
or processed or contamination occurs. Organisms include salmonella,
staphylococcal and streptococcal infections.
5.1.6 Indirect blood contamination by insect or animal bites
Rabies virus is carried in the saliva and injected when an

infected animal bites. Ticks, fleas and lice carry diseases such
as typhus and cholera. The insect will bite an infected person
and then carry the disease to a healthy person.
Try to think of diseases transmitted in each of these ways

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5.2 Pathogenic Micro-organisms
Most microbes do not produce disease. Those that do are called pathogens. Pathogenicity
is the ability of an organism to cause disease. ‘Virulence’ indicates the degree of
pathogenicity of a particular micro-organism. A virulent pathogen readily causes disease.
An infection occurs when a pathogenic microbe grows in body tissues. An infectious disease
is due to growth of a pathogenic micro-organism.
Obligate pathogens are those microbes that must cause disease in order to be transmitted
from one host to another and need to infect a host to survive.
An opportunistic pathogen is one that can be transmitted from one host to another without
having to cause disease. However, it may cause disease in situations where the hosts’
defence mechanisms are altered by factors such as stress, age or other disease, for example
when the immune system is not functioning correctly in those suffering from AIDS.
A communicable disease is transmitted from one person to another, like measles, diphtheria
and gonorrhoea, however contact with pathogenic micro-organisms does not always result
in infection. The body has a range of defence mechanisms which protect against infectious
diseases. If infection does occur, symptoms characteristic of the inflammatory reaction
usually result: redness, heat (fever), swelling and pain.
Bacteria are found just about everywhere, but only a few cause diseases. Look in section 5.1
of the British National Formulary (Table 1 - Summary of antibacterial therapy) for a list of
infections caused by bacteria. You will find drug treatment there as well.
Micro-organisms cause many types of disease and we cannot list them all here. In the
following sections are some examples of infectious diseases important because they are
particularly common or particularly serious.
5.2.1 Diseases causes by bacteria
Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis.

Years ago, this disease used to be called ‘consumption’ and resulted in many deaths.
Problems are usually seen in the respiratory tract, although it can affect other areas.
Transmission occurs by water droplets in the air. Infection with the disease has been
controlled in this country through the BCG immunisation programme which used to be given
at about age 13, before children leave school. Currently the BCG immunisation programme
targets those most likely to contract the disease, see the BNF chapter 14 for a list of
considered at risk. Antibiotic medicines are also available to treat the infection. However,
the number of cases of tuberculosis does now seem to be rising again.

40
It is more often seen in immigrant populations, particularly amongst those from the Indian
subcontinent. And there is also an increasing problem of the bacteria becoming resistant to
antibiotic treatment.
An adult with tuberculosis might describe having 'flu that never cleared up'. Cough is an on-
going symptom with increasing tiredness, weight loss and occasional blood in the sputum.
As well as the immigrant population, those living in very poor conditions, living on the
streets or in hostels, are more likely to be infected. Although antibiotic therapy is available,
treatment requires regular dosage over many months. Many people do not stay on the
course and this is a real problem if trying to eradicate tuberculosis. Stopping treatment
early can also lead to the development of strains of the TB mycobacterium which are
resistant to antibiotics.
Whooping cough (pertussis) is another infection caused by bacteria. It is usually seen in

children and it is highly infectious. The bacterium, Bordetella pertussis, is spread by water
droplets in the air. The disease begins with cold like symptoms for about a week. The cough
then becomes much worse with the characteristic 'whooping' noise as the child has trouble
breathing in during coughing fits. Coughing fits are more common at night, but in between
these attacks the child may seem perfectly alright. Although some antibiotics will kill these
bacteria, once the coughing fits are established antibiotics do not seem to change the course
of the disease. Whooping cough has largely been controlled by the vaccination programme.
Pertussis vaccination is usually given to babies in a combined vaccine that also protects
against diphtheria and tetanus. However, fears about the safety of the vaccine have
periodically caused parents to refuse vaccination and major epidemics have followed.
Gastroenteritis (an infection of the gastro-intestinal tract) is a major problem in less

developed countries where water and food supplies are often contaminated with micro-
organisms. A range of different bacteria and viruses can cause diarrhoea, abdominal pain
and vomiting. Food poisoning which occurs rapidly (less than 6 hours) is due to chemical
toxins produced by bacteria present in the food when it is eaten e.g. staphylococcal food
poisoning. If the symptoms occur less rapidly this may be due ingestion of live bacteria
which then take some time (12-24 hours) to become established and replicate (e.g.
salmonella). Other bacteria which infect the gastrointestinal tract include shigella,
campylobacter and Escherichia coli. Gastro-enteritis is also commonly caused by viruses,
rota viruses being seen particularly in young children.
Like infections of the gastrointestinal tract, infections of the upper respiratory tract are also
caused by both bacteria and viruses. As well as tuberculosis and whooping cough, bacterial
respiratory infections include diphtheria, Legionnaires disease, and streptococcal throat
infections. Various bacterial infections also occur in the urogenital system.
Vaccination against Group C Meningitis was added to the childhood immunisation
programme in 1999, with additional catch up programmes for those at particular risk.

41
5.2.2 Viral diseases
The common cold is something few of us escape once or twice each year. Colds are caused
by many viruses including rhinoviruses and adenoviruses. Colds are transmitted from
respiratory secretions in airborne droplets and by direct contact.
Influenza is another common infectious disease characterised by fever and respiratory

symptoms. There are different types of influenza virus. The ability of the virus to change its
surface antigens (glycoproteins that cause the body to the produce antibodies) means that
the body's antibody defences may fail to recognise the invading virus. So a previous
infection does not necessarily confer protection. Influenza usually starts with fever and
general aching, plus a sore throat and persistent dry cough. The fever usually goes in a few
days, but the cough and tiredness may persist.
Chickenpox and shingles are both caused by the varicella-zoster virus. Chickenpox is highly
infectious and is usually seen in children. It has a characteristic rash with red spots and
blisters. An attack of chickenpox gives immunity so repeat attacks are rare. Chickenpox is
transmitted by water droplets in the air or direct contact with the blisters. If you have had
chickenpox the varicella-zoster virus can remain dormant in parts of the body. Years later,
the virus may become active and cause shingles. Shingles normally starts with a stabbing
pain along a nerve which later is often accompanied by a skin rash which appears as a band
or raised dots, normally on one side of the body. Although the rash clears in 1-2 weeks the
pain may persist for months.
Look in the BNF and find out the names of any anti-viral drugs which can be used to treat
shingles.
Measles is caused by a type of paramyxovirus. The disease is characterised by upper

respiratory tract symptoms, cough and high fever. This is followed after a few days by a skin
rash. Transmission is by inhalation of droplets or by contact. Since the introduction of
measles vaccination, the disease has become relatively rare. In 1990 and 1991 no children
died of acute measles related illness in England and Wales. This compares with 1000 deaths
in 1949 and 90 in 1968. Rates of immunisation against measles in the UK dropped in the
early 2000s due to a now discredited scare about a possible link between the measles
vaccine and autism and this has led to new outbreaks of measles in some areas. Vaccination
rates have since increased again and teenagers who missed out on the vaccine are now
being targeted to come forward for immunisation.
The current vaccination programme includes routine vaccination against other viral diseases
such as poliomyelitis (polio), mumps and rubella (German measles). Poliomyelitis, which is
caused by polio viruses, has now been just about eradicated in the UK. German measles or
rubella is not generally serious, but because it can severely affect the unborn foetus,
vaccination is advocated, particularly for women of child bearing age.

42
Infection with the human immunodeficiency virus (HIV) can lead to AIDs, a condition in
which there is progressive deterioration of the immune system, sometimes to the extent
that the body has difficulty fighting disease and certain cancers. Although drug treatments
for HIV are now available which have greatly improved the health of many people living with
HIV and AIDs, as yet there is no vaccination or cure.
5.2.3 Diseases caused by fungi and yeasts
Ringworm and 'athlete's foot' are both skin infections caused by types of fungi. These
species of fungi which cause both ringworm and athletes foot are Microsporum,
Epidermophyton and Trichophyton. The infections transmitted by direct or indirect contact
with an affected person or animal.
Ringworm is characterised initially by a small area of red, itchy, scaly skin. The affected area
gradually becomes larger. Athlete's foot is usually seen as redness between the toes, with
the skin becoming cracked and uncomfortable. These fungi can affect any body site
including the scalp and the nails. Antifungal medicines are given to overcome these
infections.
Candida albicans is a type of yeast which causes thrush. Candida is part of the normal
microflora. It is only when overgrowth occurs that a thrush infection is seen. In very
exceptional circumstances, candida infections can spread through the whole body.
Antifungal agents are used to treat candida infections.

43
Take a Break!
You’ve reached the end of the unit! Great work so far,
however, make sure you have understood the content of
this chapter by going through the summary and attempting
the quiz questions below.
Chapter 5 Summary
• Microbes can be transferred through person to person skin contact, intimate person to
person mucus contact, direct blood contamination, dust or droplets in the air, food or
water contamination, and insect or animal bites.
• A pathogen is a microbe which causes disease. Obligate pathogens must live inside a
host and cause a disease in order to survive, whereas opportunistic pathogens can be
transmitted from one host to another without having to cause disease, however may
cause disease in situations where a host’s immune system is not functioning correctly.
• Bacterial pathogens cause diseases such as tuberculosis, whooping cough and gastro-
enteritis.
• Viral pathogens cause diseases such as the common cold, influenza, chicken pox,
measles and HIV.
• Fungal pathogens cause diseases such as ring worm, athlete’s foot and thrush.

1. What is a pathogen?
a) A microbe which does not cause disease
b) A microbe which causes disease
c) A microbe which only causes disease in the elderly
2. Which of the following diseases is caused through droplets in the air?

a) HIV
b) Gastro-enteritis
c) Tuberculosis
3. Which of the following diseases is caused through sexual contact and blood
contamination?
a) Gastro-enteritis
b) HIV
c) Whooping cough

44
4. Thrush is caused by what type of microbe?

a) Bacteria
b) Virus
c) Fungi
5. What is an obligate pathogen?

a) A microbe that needs to cause disease in order to be transmitted from one host to
another
b) A microbe that can travel between hosts without causing disease, but may cause disease
in some susceptible patients
c) A pathogen which will always cause death
1.b; 2.c; 3.b; 4.c; 5.a


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MODULE 5 MICROBIOLOGY FOR PHARMACY

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Micro-organisms will either reproduce themselves through asexual or sexual reproduction

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Chapter 1 - Types of Micro-organisms

By the end of this chapter you will be able to:

1.1.1 Bacterial Structure

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Why do you think bacterial spores make it difficult to sterilise things?

1.1.2 Classification of Bacteria

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1.1.3 Growth and Reproduction of Bacteria – Binary Fission

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Some common mould genera are Penicillium, Aspergillus, and Rhizopus.

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1.4.1 Virus Structure

Viruses are made up of a core of

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1.4.2 Classification of Viruses

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Chapter 1 Quiz - Test Yourself

2. Which of the following are prokaryotes?

4. A bacteria will have a...

5. A mould will have a....

6. A bacterial cell replicates by a process called what?

7. A eukaryotic cell DOES NOT have...

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8. A virus can have...

9. What type of virus contains reverse transcriptase?

11. A bacterial spore is what?

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Chapter 2 - Looking at Micro-organisms

By the end of this chapter you will be able to:

2.1 Light Microscope

Because of their very small size micro-organisms can only be seen

Would you be able to see viruses with this type of microscope?

A light microscope can be used to

2.2 Electron Microscope

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2.3 Other Forms of Microscopy

2.3.1 Phase Contrast

2.3.2 Dark Ground

Dark ground microscopy is a technique whereby the

Dark ground microscopy can be used on live and unstained

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Chapter 2 Quiz - Test Yourself

2. Which type of microscopy has the highest magnification?

3. Which type of microscopy produces high contrast images of specimens of which it is

5. A disadvantage of electron microscopy is....

1.b; 2.a; 3.c; 4.b; 5.a

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Chapter 3 - Growth of Micro-organisms

By the end of this chapter you will be able to: