Biotechnology
Unit 2: Microbiological Techniques
Student Materials
[HIGHER]
Margot McKerrell
© Learning and Teaching Scotland
abc
The Scottish Qualifications Authority regularly reviews
the arrangements for National Qualifications. Users of all
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or others, are reminded that it is their responsibility to
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Acknowledgement
Learning and Teaching Scotland gratefully acknowledge this contribution to the National
Qualifications support programme for Biotechnology. The advice of Jim Stafford is
acknowledged with thanks.
First published 2004
© Learning and Teaching Scotland 2004
This publication may be reproduced in whole or in part for educational purposes by
educational establishments in Scotland provided that no profit accrues at any stage.
ISBN 1 84399 058 X
© Learning and Teaching Scotland
CONTENTS
Introduction
5
Section 1:
Growth limitation and sterilisation techniques
7
Section 2:
Culturing techniques
15
Section 3:
Identification of micro-organisms
37
Bibliography
41
Appendix:
45
Advice for Outcome 2
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INTRODUCTION
This unit introduces you to some of the techniques that are used to
study micro-organisms. A micro-organism is any small organism that
cannot be clearly seen without the help of a microscope. The microorganisms that you will use to carry out the study of microbiological
techniques include bacteria, fungi and viruses. Many biotechnology
processes rely on the use of micro-organisms and so it is important that
you know how to work safely with them. That is why this unit is included
in Higher Biotechnology.
These student notes will provide you with the knowledge and
understanding that you will need to carry out microbiological
techniques.
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GROWTH LIMITATION AND STERILISATION TECHNIQUES
SECTION 1
Growth limitation and sterilisation techniques
Some micro-organisms can be harmful to humans as they cause disease.
For this reason, techniques have been developed to control the
unwanted growth and spread of micro-organisms. It is essential that you
have an understanding of these techniques before you start to culture
micro-organisms in the laboratory. This is to ensure that you safely
culture and contain the micro-organism that you are interested in and
do not contaminate yourself, others or the environment.
Sterilisation and disinfection
Sterilisation is a process that kills all micro-organisms, including
endospores, within a material or object. (An endospore is a dormant
structure formed from a bacterial cell that can survive extremely adverse
conditions, including high temperatures. It can germinate into a
bacterial cell if growth conditions become favourable.)
Any physical or chemical agent that kills micro-organisms is said to be
biocidal.
There are several techniques used to sterilise materials and objects and
these are discussed below.
Autoclaving is a technique that kills micro-organisms using pressurised
steam. Under normal atmospheric pressure the highest temperature
that steam can reach is 100°C. While this temperature will kill many
micro-organisms, it is too low a temperature to kill some endospores. In
order to increase the temperature of steam above 100°C, it is
pressurised in a closed container called an autoclave. Micro-organisms
(including their endospores) are killed at these high temperatures
because their enzymes and proteins are denatured, so they are no
longer viable.
In a laboratory, items are sterilised in autoclaves at 121°C for 15 minutes
or at 126°C for 10 minutes. The times mentioned here are average
sterilisation times. The time that objects and materials are autoclaved
depends on their size as large objects take longer to sterilise. Also, the
autoclave should be loosely packed so that the steam can circulate
around the objects and so heat them to the correct temperature. If the
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correct temperature or time is not observed, then the items inside the
autoclave cannot be guaranteed to be sterile. To ensure that the correct
autoclaving procedure has been carried out, autoclave monitors such as
Browne’s tubes and test strips, are added to the autoclave with the items
to be sterilised and then checked at the end of the procedure. In
general, these monitors change colour to show that sterilisation has
taken place; for example, Browne’s tubes change from red to black
when they have been sterilised correctly.
The autoclave is used to sterilise objects and materials that are heatresistant such as glassware, cloth, rubber, metallic instruments, liquids,
paper and heat-resistant plastic. In the laboratory in your school or
college, you may use an autoclave to sterilise culture media, scalpels and
glassware such as test tubes and conical flasks.
Another technique that is used to sterilise materials and objects is the
use of dry heat. Dry heat sterilisation takes place in a dry oven that has
electrical coils that radiate heat. Dry heat kills micro-organisms by
dehydrating them and denaturing their proteins. Higher temperatures
are needed when using dry heat compared to using steam. In general,
items are sterilised at 160°C for two hours.
Dry heat is used to sterilise items that may be adversely affected by
steam, such as powders, oils, glass pipettes and metal instruments that
may corrode if exposed to moisture. Dry heat is not suitable for plastic,
cotton, paper or for solutions that would boil and dry out, such as
culture media.
Some liquids cannot be sterilised by autoclaving or in dry-heat ovens
because the temperatures used in these techniques cause the
components in the liquids to denature. Heat-sensitive liquids can be
sterilised using filtration.
One type of filtration uses a filter that is composed of a mass of fibres.
When liquid is passed through this type of filter, any micro-organisms
present in the liquid adsorb (stick) to the fibres. Another type of
filtration uses a filter that has pores (holes) large enough for liquid to
pass through but too small to let micro-organisms through, so they
become trapped on the filter. These filters are known as membrane
filters.
Filtration is used to sterilise solutions containing antibiotics, enzymes
and glucose. It is also used to sterilise air, for example before it is
pumped into a fermenter.
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Another technique used for sterilisation is gamma irradiation. Items to
be sterilised in this way are placed in a machine that emits gamma rays
and these items are exposed to the gamma rays for a specific time.
Gamma rays kill micro-organisms by causing lethal mutations in the DNA
that cannot be repaired.
Gamma irradiation is used to sterilise plastics such as Petri dishes and
syringes, and surgical gloves.
Disinfection is when a chemical agent (disinfectant) is used to destroy
micro-organisms but not endospores. Disinfectants do not normally
achieve sterility because they do not always kill all micro-organisms
present. However, they do reduce the number of micro-organisms to
safer levels. In general, disinfectants are used on non-living objects
because they are toxic to living tissues. Disinfectants that can be applied
to living tissue are known as antiseptics.
Some disinfectants are biocidal (look back to page 7 to remind yourself
what this term means). Other disinfectants are biostatic. This means
that micro-organisms are temporarily prevented from reproducing but
they are not killed. When this type of disinfectant is removed, the microorganism can start to increase in numbers again.
When using disinfectants, it is important to ensure that they are used at
the correct concentration and that they are left to work for the correct
length of time. In general, the higher the concentration of a
disinfectant, then the faster the micro-organisms are killed. This is
shown in the graph below:
Figure 1
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Although high concentrations of disinfectant may be more effective at
destroying larger numbers of micro-organisms more quickly, high
concentrations of disinfectant tend to be more toxic and expensive. For
these reasons disinfectants are generally used at the minimum
concentration that effectively destroys micro-organisms.
Apart from concentration, another factor that affects disinfectants is
time. Most disinfectants require time to act on the micro-organisms.
Also, the more micro-organisms that are present, then the longer the
time needed to destroy them.
Many laboratory disinfectants need to be prepared before they can be
used. Some liquid disinfectants are diluted by putting a small volume of
the disinfectant into a large volume of solvent. Powdered disinfectants
are weighed out, then dissolved in an appropriate volume of solvent. In
general the manufacturer of the disinfectant provides clear guidelines
regarding the most appropriate concentration that should be used and
how long it will remain active.
In the laboratory, disinfectants are used for a variety of purposes such as
swabbing a bench before and after use, for the sterilisation of surfaces,
and for the disposal of used instruments such as Pasteur pipettes.
There are many disinfectants available and some of the more common
ones are discussed below:
Chlorine
• Chlorine kills bacteria, fungi, viruses and endospores.
• It is an oxidising agent and works by denaturing proteins.
• It is used in the treatment of drinking water.
• Chlorine-based disinfectants are used in the home and laboratory for
disinfecting surfaces.
Phenolics
• Phenolics kill bacteria, fungi and some viruses.
• They work by dissolving cell membranes and denaturing proteins.
• They are found in carbolic soaps and some laboratory disinfectants.
Alcohol
• Alcohol kills bacteria, fungi and some viruses (if left long enough).
• It works by dissolving cell membranes.
• It is used in the laboratory for swabbing benches and disinfecting
instruments such as scalpels.
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Multi-oxidising detergents
• These destroy a wide variety of micro-organisms.
• They work by denaturing proteins.
• They are used in the laboratory for a variety of purposes such as
swabbing benches and in disposal containers for used glass pipettes.
Risk assessment and coping with spillages
When working with micro-organisms, it is extremely important that you
are aware of the possible hazards and risks associated with them. A
hazard is the danger or harm that a micro-organism may cause to you. A
risk is the probability or likelihood that you will be harmed by the
micro-organism.
Therefore, before working with micro-organisms, a risk assessment is
carried out. To do this, you look at the procedures that you are
intending to carry out using a micro-organism and then assess the
potential risks associated with these procedures. Risk assessments
generally fall into one of three main categories: simple, generic and
novel risk assessments.
Simple risk assessments involve the use of micro-organisms and
procedures that pose a familiar hazard and there are well-known control
measures available to minimise the risks associated with them.
Generic risk assessments involve the use of an authoritative source of
advice or code of practice for the safe handling of a micro-organism
when using a particular procedure.
Novel risk assessment is the procedure adopted when you come across a
hazard that is unfamiliar to you and is not covered by the generic code
of practice. In this case, you must research the potential risks from first
principles.
After you have identified the hazards and potential risks associated with
working with micro-organisms, it is important to use appropriate control
measures to reduce the risks to a minimum. Some control measures that
are taken are outlined below:
• Choice of micro-organism. The most appropriate micro-organism
with the fewest hazards associated with it is chosen. Micro-organisms
should be obtained from known, reputable sources to ensure that
you are working with a pure culture. The micro-organism should be
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stored in the correct conditions and be properly labelled with the
name of the micro-organism and the date that it was obtained. A log
book/record with details such as culture number and when it has
been subcultured should be kept.
• Media selection. Media that are most suitable for the micro-organism
should be used, for example selective media rather than generalpurpose media should be used. This helps to prevent the growth of
unwanted micro-organisms. Media likely to encourage the growth of
pathogenic (disease-causing) micro-organisms should be avoided.
(You will find out more about different types of media later on in
these notes.)
• Culture methods. The micro-organism should be grown in the
correct conditions such as temperature, pH and with the correct
oxygen requirements. For example, do not grow aerobic bacteria in
anaerobic conditions as anaerobic pathogens may grow instead. Also,
plastic culture vessels are preferable to glass as they are less likely to
break if dropped.
• Choose appropriate handling procedures. For example, try to
prevent aerosol formation. Aerosols are water droplets containing
micro-organisms that are released into the atmosphere. They can
remain in the air for long periods and be a source of contamination.
There are many ways to prevent aerosol formation; for example, do
not put a wire loop with lots of culture on it directly into the hottest
part of the Bunsen burner. Instead, introduce the loop gradually into
the flame. Also, aerosols are created when containers of liquid are
opened, so take care when opening containers of micro-organisms.
Other handling procedures that should be considered are the scale of
the operation, the degree of containment and the likelihood of
contamination. For all of these you need to look closely at your
procedures and ensure that you have sufficient resources to cope
with them.
• Protective equipment. A laboratory coat worn correctly protects
your everyday clothes from contamination. Lab coats should be
removed before leaving the lab to prevent taking contamination
outside of the laboratory. Eye protection should also be worn to
protect your eyes from air-borne contamination and chemicals.
Gloves can be worn to protect your hands when handling microorganisms and can then be disposed of after use. If gloves are not
worn (and they are not always needed!), then cuts should be covered
with waterproof plasters. Hands should be washed thoroughly with
anti-microbial soap before and after handling micro-organisms.
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Smoking, eating and drinking are not allowed in microbiology
laboratories as your mouth and gastro-intestinal tract could be
contaminated.
Treatment of small-scale spillages of broth
Accidents can happen and it is possible that you may spill a live culture
of micro-organisms when you are working in the laboratory. If this
happens, then it is important to prevent contamination of yourself,
others in the laboratory and the laboratory itself. There are several steps
that should be taken to treat a small-scale spillage. Firstly, the person
disposing of the spill must wear a lab coat, disposable gloves and eye
protection. The spill is covered with paper towels and disinfectant is
poured around and over the towels to prevent aerosol formation. This is
left for at least 10 minutes to ensure that the disinfectant has had
enough time to work; then the paper towels are carefully put into a
disposal bag and autoclaved. The gloves should be autoclaved too as
they may be a source of contamination. If a culture vessel was broken in
the accident, it should be put into a solid container or put into two
disposal bags and autoclaved.
Containment of large-scale spillages
Large-scale spillages may happen in industrial processes using large
fermenters. There are a number of precautions that are taken to contain
spillages from fermenters, so preventing contamination of the
environment. In rooms where there are large fermenters, drains do not
directly lead to the main sewerage system. Instead they lead to sumps
(large containers) where spillages can be decontaminated before being
introduced into the main sewerage system. Floors and walls are sealed
to ensure that they are waterproof. Doorways are positioned above
ground level so that spillages do not leak out into the environment.
You have now finished the first part of this unit and you should be
familiar with the procedures used to prevent the unwanted growth of
micro-organisms. You should be familiar with methods of sterilisation
and disinfection; know the difference between a hazard and a risk; be
aware of control procedures that you can carry out to minimise the
risks of working with micro-organisms; know how to deal with smallscale spillages; and how large-scale spillages of broth are contained.
You are now ready to be introduced to some of the techniques that are
used to grow micro-organisms.
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CULTURING TECHNIQUES
SECTION 2
Culturing techniques
This section of the unit introduces you to the theory behind the
techniques that are used to culture micro-organisms. However, you
should remember that this unit is a ‘hands-on’ practical unit and you will
be given the opportunity to carry out many of the techniques in the
laboratory so that you will become competent in the safe handling of
micro-organisms. Your tutor will provide you with the protocols and
methods that you need to carry out the techniques safely.
While working with living micro-organisms you must develop and use
good working/laboratory practices. These practices are important for
several reasons:
• to ensure that you do not contaminate yourself or others in the
laboratory, or contaminate the laboratory itself
• to avoid contaminating the cultures with which you are working
• to prevent accidentally taking micro-organisms out of the laboratory.
Aseptic technique is the name given to all the procedures that are used
when working with micro-organisms to prevent contamination. Some of
the procedures that are used and the reasons for them are described
below.
Firstly, it is essential that you prepare yourself in readiness for carrying
out practical work with micro-organisms. Long hair must be tied back,
hands must be washed with soap and water, cuts covered with
waterproof plasters (alternatively plastic gloves can be used) and
personal protective equipment such as a lab coat and eye protection
must be worn. You must ensure that the sleeves of your lab coat are
rolled down and that it is buttoned up to protect your normal clothes
from accidental spillages.
Next, you must prepare your work space. Work benches used for
microbiology must be smooth and non-absorbent so that they do not
become contaminated if there is a spillage. If necessary, benches can be
made suitable for microbiology work by covering them with nonabsorbent material such as benchcote. Before starting work, bench
surfaces are always disinfected to reduce the possibility of contaminating
the cultures with which you are working. It is good practice to have a
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container of disinfectant close to your work space so that apparatus,
such as pipettes, can be disposed of quickly and easily without
contaminating the environment.
A Bunsen burner is placed on the work space. When lit, it provides an
updraught that carries air away from the work space, so reducing
contamination. It is also used to flame-sterilise equipment such as wire
loops and the necks of test tubes and flasks before and after the transfer
of micro-organisms.
At the completion of work, your work space must be disinfected.
Unwanted cultures and apparatus contaminated with micro-organisms
must be disposed of properly. Unwanted cultures are autoclaved and
then disposed of in the bin, in the case of used agar plates in Petri
dishes, or down the sink in the case of liquid broth cultures.
Contaminated items of apparatus are first put into disposal containers of
disinfectant, then autoclaved.
Media preparation and sterilisation
Micro-organisms are grown in culture media that contain a range of
nutrients necessary for growth. In a laboratory, culture media is
contained within Petri dishes, test tubes, bottles and flasks.
Prior to being used, all containers are labelled with the type of culture
media that they contain and with the date when the culture media was
prepared. They may also be labelled with the initials of the person who
prepared them. Remember, a Petri dish is labelled on the bottom plate,
never on the lid.
Culture media is sterilised in an autoclave for the appropriate length of
time, depending on the volume of media that is to be sterilised. When
sterilising media in an autoclave, it is important that the containers are
not filled to capacity, otherwise they may boil over in the autoclave or
the container may crack or break.
Sterile culture media is poured from one container to another using the
appropriate aseptic technique. This includes flaming the neck of the
container before and after the transfer of culture media from one
container to another.
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The preparation of plates
Culture media can be solidified by the addition of agar. Culture media
containing agar becomes molten when it is autoclaved, then as it cools
down to 42°C, the agar solidifies. Solid culture media in a Petri dish is
known as an agar plate.
Agar plates are prepared by adding water to the appropriate quantity of
powdered culture media (follow the manufacturer’s instructions),
autoclaving, cooling to about 50°C, and then pouring aseptically into
Petri dishes. When the agar has solidified to a flat, smooth surface, the
agar plates are inverted and allowed to dry. This prevents condensation
forming on the surface of the agar.
The preparation of slopes
Solid culture media can also be contained within a universal or
McCartney bottle (this is a bottle with a flat bottom and a screw cap that
holds about 20cm3 liquid). These bottles are often used to prepare agar
slopes. The bottle is filled with about 15cm3 molten, sterilised agar
media and kept in a tilted position while the agar solidifies, thus forming
a slope of agar.
The preparation of broths
Liquid culture media is known as a broth and is contained within test
tubes or conical flasks. These containers are always loosely stoppered to
prevent contaminating micro-organisms from gaining entry, but allowing
air to enter.
Suitability of media for inoculation
After culture media has been prepared, it is always examined for its
suitability for inoculation.
Culture media is examined to ensure that it is free from contamination
with no visible signs of growth on solid or in liquid media. Agar plates
should be dry, flat and smooth. Slopes should be flat and smooth.
Types of media
Culture media contains the nutrients in the correct proportions needed
by micro-organisms for growth.
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The nutrients needed by micro-organisms for growth, why the nutrients
are needed and the sources of these nutrients in culture media are
shown in the following table:
Nutrient
Why the nutrient is
needed by the
micro-organism
Source of the nutrient
in culture media
Nitrogen
• To make proteins
and nucleic acids
• Amino acids and
proteins
• Ammonium ions
• Nitrate ions
Carbon
• To make carbohydrate,
proteins, nucleic
acids and lipids
• As a source of energy
• Sugars and amino
acids
• Sugars
Phosphorus
• To make nucleic acid
and certain types of
lipids
• Phosphate ions
Sulphur
• To make proteins
• Sulphate ions
In addition to nutrients, culture media contain buffers that ensure the
pH of the culture media stays the same. This is important because microorganisms grow at optimum pH values and any change in pH may affect
the growth of the micro-organism. Some micro-organisms produce acid
as they grow which could change the pH of the culture media and so
inhibit growth. Buffers help to prevent the acid produced from changing
the pH of the media.
As mentioned previously, some culture media is solidified by the
addition of agar, a polysaccharide obtained from marine algae. It does
not add nutrients to the media as few micro-organisms produce
enzymes to metabolise it. Agar is molten at high temperatures and
solidifies as it approaches 42°C.
There are many different types of culture media, but they all fall into two
main types, synthetic and complex media.
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Complex media are those in which the nutrients obtained from the
ingredients are not present in defined (known) quantities. For example,
some complex media contain peptone, which is a mix of proteins that
have been partially hydrolysed (broken down). Peptone is a source of
carbon, nitrogen and sulphur but the actual quantities may vary from
batch to batch. Other complex media contain yeast or beef extract,
which are sources of amino acids, sugars and vitamins.
Synthetic media are those in which pure chemical components are
added in known quantities. An example of a synthetic medium is Czapek
Dox that is used to culture fungi. The quantity of ingredients used to
make a volume of 1000cm3 is shown below:
Sodium nitrate
Potassium chloride
Magnesium glycerophosphate
Iron sulphate
Sucrose
Agar
20.0g
0.5g
0.5g
0.01g
0.35g
12.0g
General-purpose media are complex media that are designed to grow a
broad spectrum of micro-organisms. Nutrient broth and nutrient agar
are examples of general-purpose media that are used commonly in
laboratories for the culture of bacteria.
Selective media contain ingredients that allow the growth of some
micro-organisms in a mixture but inhibit the growth of other bacteria in
the same mixture. An example of selective media is mannitol salt agar
that is used to select for Staphylococci bacteria.
Differential media make it easy to identify colonies of one bacterium
from colonies of another bacterium on the same agar plate. An example
of differential media is MacConkey agar, which is used to identify human
intestinal micro-organisms as a test for faecal contamination (just to
confuse you, MacConkey agar is also an example of selective media!
This is explained on pages 23 and 24).
Isolating and culturing micro-organisms
To culture a micro-organism, a sample (called the inoculum) is
introduced into a culture medium that provides an environment in
which the micro-organism can multiply. The observable growth that
appears in the medium is known as a culture.
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A pure culture is one that contains a single known species or type of
micro-organism. This type of culture is most frequently used for the
study of micro-organisms in the laboratory.
A mixed culture is one that has two or more known, easily
differentiated species of micro-organisms.
A contaminated culture is one that was once pure or mixed but has
since had contaminants (unwanted and unknown micro-organisms)
introduced into it.
When culturing micro-organisms in the laboratory, it is important to use
aseptic techniques at all times to reduce the risk of producing a
contaminated culture.
Micro-organisms are transferred from one culture medium to another, a
process known as sub-culturing, before incubation. The following table
shows the four sub-culturing techniques that can be carried out, the
culture medium that the micro-organisms are taken from, and the
culture medium to which the micro-organisms are transferred:
Sub-culturing
technique
Culture medium
that micro-organisms
are taken from
Culture medium that
the micro-organisms
are transferred to
Solid to solid
Agar plate
or
Agar slope
Agar plate
or
Agar slope
Solid to liquid
Agar plate
or
Agar slope
Liquid broth
Liquid broth
Agar plate
or
Agar slope
Liquid broth
Liquid broth
Liquid to solid
Liquid to liquid
The inoculum is transferred using either a loop or pipette. A loop can
be used in all four sub-culture techniques. A pipette can be used to
transfer liquid to solid and liquid to liquid.
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When transferring micro-organisms from one medium to another, strict
aseptic technique must be followed such as having a Bunsen burner lit
on the work space, flaming the loop before and after each transfer, using
sterile pipettes, and flaming the neck of containers with liquid broth
before and after transfer of micro-organisms.
One method for transferring micro-organisms onto an agar plate is
called the streak plate technique. Micro-organisms can be transferred
from liquid broth, from a slope or from an agar plate onto an agar plate
by this method. Figure 2 shows the steps involved in streak plating.
Figure 2
Firstly a loop-full of micro-organism is smeared across the edge of an
agar plate (1). The loop is sterilised by flaming, and cooled. The loop is
used to make several streaks (usually three) through the first set of
streaks (2). Again the loop is sterilised and cooled and the process
repeated a further twice (3) and (4). The number of micro-organisms
decreases with each set of streaks, so that by the final set of streaks,
individual colonies are produced (4) after the agar plate has been
incubated.
Growth of micro-organisms
Following transfer into a growth medium, micro-organisms will
reproduce and increase in number to produce a culture, providing they
are given the correct growth conditions. While the culture medium
provides the nutrients necessary for growth, micro-organisms also
require the correct temperature, pH, level of oxygen and salt for
maximum growth.
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All micro-organisms have their own optimum temperature at which they
grow best. Some micro-organisms, such as those isolated from soil, have
an optimum pH in the range between 0°C and 25°C. Other microorganisms, such as those that cause disease in mammals, have an
optimum temperature in the range from 20°C to 40°C. There are some
micro-organisms, which are found naturally in compost, that grow best
in the temperature range from 45°C to 60°C.
Micro-organisms also have an optimum pH for maximum growth. In
general, fungi are acid tolerant and grow best in the pH range 5 to 6.
Bacteria prefer to be grown at pH 6.5 to pH 7.5.
Different micro-organisms have different requirements for oxygen.
Obligate aerobes must be grown in the presence of oxygen whereas
obligate anaerobes must be grown in the absence of oxygen.
Facultative anaerobes can grow in the presence and absence of
oxygen.
Micro-organisms that live naturally in a salt environment, such as those
that live in salt lakes and seas, must be provided with culture media that
is supplemented with salt. Some micro-organisms will not grow unless
they are provided with a minimum of 9% salt (that is 9 grams of salt per
100cm 3 medium).
Assuming that the micro-organisms are provided with the correct
nutrients and growth conditions, growth of the micro-organism in the
culture media will occur. Growth is observed in liquid cultures when the
broth turns cloudy or turbid. Growth is observed on agar plates by the
appearance of colonies.
It is difficult to be sure that a liquid culture contains a pure culture of
micro-organisms and that it has not been contaminated. It is easier to
observe if an agar plate contains a pure, mixed or contaminated culture.
(Look back to the beginning of this part of the unit to remind yourself of
the differences between a pure, mixed and contaminated culture). If a
culture is pure, all the colonies on the plate are identical in colour,
shape and size. In a mixed culture there are two or more (depending on
the number of different species present) types of colonies with different
colour, size and shape.
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Pure cultures
In laboratories, pure cultures are normally used and there are many
reasons for this:
• When studying the characteristics of a micro-organism, other microorganisms should not be present. Otherwise you will not know for
certain that it is the micro-organism being studied that is carrying out
the reaction.
• Contaminating micro-organisms use up nutrients in the medium, so
there is less nutrient and so less growth of the actual micro-organism
being studied.
• Contaminating micro-organisms may produce substances that prevent
the growth of the micro-organism being studied.
Pure cultures are also used in industrial fermentation where a
commercial product is being made. Contamination in a fermentation
process may lead to reduced product being formed (if there is less
growth of the micro-organism carrying out the fermentation), or the
product may be impure because of the presence of substances produced
by the contaminating micro-organism.
There are a number of ways that pure cultures can be obtained from a
mixed culture, for example by using selective and differential media and
by exploiting discrete growth characteristics of the micro-organisms.
A selective medium contains one or more ingredients that prevent the
growth of certain micro-organisms but not others. These media are
important in the isolation of one type of micro-organism from samples
containing lots of types of micro-organisms such as those found in
faeces, saliva, water and soil.
Mannitol salt agar is a selective medium that contains high
concentrations of salt. This prevents the growth of most human
pathogens (bacteria that cause disease) but the bacterium
Staphylococcus grows in this medium, so it is used to select for this
micro-organism.
MacConkey agar is a selective medium that contains bile salts. This
medium can be used to select for bacteria (such as E. coli) that normally
grow in the human intestines as they can grow in the presence of bile
salts.
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A differential medium is designed to show visible differences between
micro-organisms such as different bacteria producing different colours
of colonies.
These differences arise from the ingredients of the media and the way
that different micro-organisms react to them. Some differential media
contain dyes that change colour if the pH changes.
MacConkey agar is an example of a differential medium that contains a
dye that is yellow if the pH is neutral and red if the pH is acidic. E.coli
bacteria produce acid when grown on this agar and so E.coli colonies
are red. Salmonella bacteria do not produce acid on this agar, so its
colonies are a natural, off-white colour.
In addition to using selective and differential media, discrete growth
characteristics (such as temperature optimum, pH optimum, and oxygen
requirements) of individual micro-organisms can be exploited to obtain
pure cultures. For example, if a mixed culture contains aerobic and
anaerobic bacteria, then anaerobic bacteria can be isolated from the
aerobic bacteria by growing them in the absence of oxygen.
Enumerating micro-organisms
Enumerating micro-organisms means counting the number of microorganisms in a given sample. Counting micro-organisms is not always
easy because of the large number of micro-organisms that can be
present, even in a relatively small sample. For example, a small drop of
liquid culture of bacteria could contain as many as several million
bacterial cells. It is impossible to give the exact number of microorganisms that are present in a sample, so an estimate of the number of
micro-organisms is given.
A sample with many micro-organisms is diluted so that a smaller number
of micro-organisms is present, which is easier to count. The number of
micro-organisms in the diluted sample is then multiplied by the dilution
factor to give an estimate of the number of micro-organisms in the
original, undiluted sample. For example, if a sample is diluted by 10–5 (1
in 100,000) and the number of micro-organisms in the diluted sample is
25, then the original sample has 25 × 100,000 = 2.5 × 106 microorganisms in it.
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Generally, dilutions are carried out in a series of sequential steps known
as a serial dilution. This is shown in Figure 3:
Figure 3
When a 1 cm3 sample is diluted in 9 cm3, this is a 1 in 10 dilution. This is
shown in Figure 3 where 1 cm3 is taken from 1 and put into 2. When
1 cm3 is taken from 2 and diluted a further 1 in 10, the resulting dilution
in 3 is a 1 in 100 dilution (1 in 10 × 1 in 10). A 1 in 100 dilution is also
known as a 10–2 dilution.
When a 0.1 cm3 sample is diluted in 9.9 cm3, this is a 1 in 100 dilution.
This is shown in Figure 3 when 0.1 cm3 from 3 is put into 4. This is a 1
in 100 dilution of a sample already diluted 1 in 100. The sample in 4 has
now been diluted 1 in 10,000 (1 in 100 × 1 in 100). A 1 in 10,000
dilution is also known as a 10–4 dilution. When 0.1 cm3 is taken from 4
and put into 5, this is a 1 in 100 dilution of a sample already diluted 1 in
10,000. The sample in 5 has now been diluted 1 in 1,000,000 (1 in 100 ×
1 in 10,000). A 1 in 1,000,000 dilution is also known as a 10–6 dilution.
When enumerating micro-organisms, there are two counts that can be
made: a total count and a viable count.
A total count is a count of all living and dead micro-organisms in the
sample.
A viable count is a count of the living micro-organisms only.
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Direct methods of enumeration involve counting the number of microorganisms directly, for example by counting the number of colonies on
an agar plate or by using a microscope to count the number of cells
observed.
Indirect counts can be made by growing micro-organisms in liquid
broth. As micro-organisms grow, the broth becomes cloudy or turbid.
The turbidity is measured using a device such as a colorimeter or a
spectrophotometer.
Direct counting of micro-organisms using a haemocytometer
A haemocytometer is a counting chamber as shown in Figure 4.
Figure 4
It is a thick glass slide containing a well in the central section. On the
bottom of the well a grid is etched containing squares of known area.
Each square is 0.04mm 2. A coverslip is placed over this well forming a
chamber of known depth (0.1mm). Thus, the volume of each square is
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known (0.04 × 0.1= 0.004mm3). The sample to be counted is placed in
the well by placing a drop of the sample at the edge of the coverslip so
that it runs into the well and over the grid. The haemocytometer is then
viewed using a microscope. The number of micro-organisms in several
squares is counted and an average number of micro-organisms is
obtained. This number of micro-organisms is then used to calculate the
number of micro-organisms in the original sample.
Figure 5 shows bacterial cells observed on one large square of the grid.
Figure 5
From this diagram, you can see that micro-organisms sometimes lie
between one large square and the next. It is standard practice to count
micro-organisms on the top and right-hand side of the square, but to
ignore the bottom and left-hand side.
From Figure 5:
5 micro-organisms are counted in one large square,
so there are 5 micro-organisms in 0.004mm3 sample
so, in 1mm3 there are
5 × 1
= 1250 micro-organisms per mm3
0.004
so, in 1cm3, there are 1250 × 1000 = 1.25 × 106 microorganisms
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If the original sample was diluted, the dilution factor must be taken into
account, for example if the original sample was diluted 1 in 1000 (10–3),
then the original sample would contain 1.25 × 10 6 × 1000 = 1.25 × 109
micro-organisms per cm 3.
Indirect counting of micro-organisms using a colorimeter or
spectrophotometer
The total number of micro-organisms can also be estimated by indirect
counting using a colorimeter or spectrophotometer. The number of
micro-organisms in a suspension can be estimated from a turbidity
reading on a colorimeter or spectrophotometer. These instruments
measure the cloudiness (turbidity) of a suspension of micro-organisms.
The more micro-organisms there are in the suspension, the higher the
turbidity, so the higher the optical density reading from the
instruments.
The total number of micro-organisms in a suspension can be worked out
from the turbidity reading if a standard curve is available. A standard
curve is generated by obtaining the optical density of micro-organisms of
known number in a colorimeter or spectrophotometer. This data is
plotted to form a standard curve. An example of a standard curve is
shown in the graph on the next page (Figure 6).
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Figure 6
The number of micro-organisms in an unknown sample can be estimated
if its optical density is obtained. It is then a simple matter of reading the
total count from the standard curve. From Figure 6, it can be seen that if
the optical density is 0.5, then the total count is 2.5 × 106 microorganisms per cm3.
Viable count of micro-organisms in a sample
A viable count is the number of living micro-organisms in a sample. The
commonest method of doing this is to use a plate count.
Firstly, a serial dilution is carried out on the sample. The diluted
samples are then plated onto an agar plate.
After incubation, colonies of micro-organisms appear on the agar plates.
Plates with between 20 and 200 colonies are generally counted.
The number of colonies observed on suitable plates is then multiplied by
the dilution factor to estimate the actual number of colonies in the
sample.
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Figure 7 shows the dilutions that were carried out on a sample of
bacteria and the number of colonies observed on the plates at each
dilution:
Figure 7
Plates B and C would be counted as they contain between 20 and 200
colonies.
For Plate B,
180 colonies grew using 0.1cm 3 of the 10 –2 dilution.
So, there are 1.8 × 105 colonies in 1cm3 sample. (180 × 100 × 10)
For Plate C
22 colonies grew using 0.1cm3 of the 10–3 dilution.
So, there are 2.2 × 105 colonies in 1cm3 sample. (22 × 1000 × 10)
The numbers obtained from Plates B and C are averaged to give an
estimate of the number of micro-organisms in the original sample, and
this works out to be 2.0 × 105 micro-organisms in 1cm3 sample.
Generally, more than one plate of each dilution is cultured and counted
to increase the reliability of the estimate.
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Plaque assay to enumerate bacteriophage numbers
This technique is used to estimate the number of bacteriophage in a
sample. Bacteriophage grow only when they have infected bacterial
cells. After growing within a cell, bacteriophage break open (lyse) the
bacterial cell. If bacteriophage-infected bacterial cells are grown on agar
plates, bacterial cells that have been lysed are observed on the plate
because the agar is exposed as a clearing (known as a plaque) on the
plate.
This is shown in Figure 8:
Figure 8
A plaque assay is carried out by doing a serial dilution of the
bacteriophage and mixing each dilution with bacteria. A known volume
of bacteriophage and bacteria is mixed with molten agar, and poured
onto an agar plate. This is then incubated at the required temperature.
Plaques (areas of clearing) are counted. Each plaque is assumed to have
arisen from a single bacteriophage and so the original number of
bacteriophage can be estimated in the same way as described for
counting colonies on a plate (page 30).
Preparing a bacterial lawn
When a large number of bacteria are inoculated onto an agar plate, they
do not grow as separate colonies. Instead, the colonies grow into each
other and the entire surface of the plate becomes uniformly covered
with bacteria. This is known as a bacterial lawn.
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There are two ways of preparing a bacterial lawn:
• by spreading a small volume (0.1cm3) of a dense culture of bacteria
over the surface of the agar plate
• by mixing a larger volume (1 cm3) of bacterial culture with molten
agar, then pouring the mixture into a Petri dish.
Bacterial lawns are used when testing the sensitivity of a bacteria against
a range of antibiotics. After the agar has been inoculated, discs
impregnated with antibiotic are placed on top of the agar and the agar
plate is incubated at the correct temperature for an appropriate length
of time. Bacteria that are sensitive to an antibiotic do not grow around
the disc and a clear area is observed. Bacteria that are resistant to an
antibiotic can grow around the disc. An example of this is shown in
Figure 9:
Figure 9
This figure shows that this bacteria is sensitive to penicillin but resistant
to tetracycline.
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Tissue culture
It is possible to culture isolated plant tissue in the laboratory using
techniques similar to those for culturing micro-organisms. When
culturing plant tissues, strict aseptic technique is needed to maintain
sterile conditions and to prevent contamination of the plant cells by
micro-organisms.
Plant tissue culture can be used to produce thousands of identical
plants. It allows commercial plant growers to produce clones of plants
such as pineapple, rose, orchid and palm oil in a relatively short period
of time. Another advantage of producing plants in this way is that they
are pathogen-free (this means that they are not diseased).
One method by which plants are micropropagated is by isolating a piece
of plant tissue, called an explant, and culturing it under sterile
conditions, either in agar or liquid medium. If the explant is given the
correct nutrients, vitamins and plant growth substances, the cells of the
explant first divide into a mass of undifferentiated cells, called the
callus, then the cells differentiate and finally they develop into a plant.
Plants can also be micropropagated from the apical meristem of a plant.
The apical meristem is the tissue found at the tip of the shoots. The cells
in this tissue are actively dividing and produce new growth of stems.
These cells are normally free from pathogenic (disease causing) microorganisms, so plants regenerated from them are pathogen-free.
The meristem is removed from the plant and sterilised with disinfectant.
It is then placed on agar containing nutrients until a shoot develops.
The shoot is placed in agar containing plant growth substances that
induce the development of roots. When roots have developed, the
plantlets are planted in sterile compost.
The nutrient medium used in micropropagation contains a variety of
substances essential for growth. Some of the substances found in the
nutrient medium are described below:
• It contains a source of carbon. A callus does not have chlorophyll,
the pigment which is involved in photosynthesis (the process that
produces sugar in a plant). Thus, the nutrient medium contains a
source of carbon, usually in the form of a sugar. The sugar is used by
the callus in aerobic respiration, the process that provides the plant
with energy in the form of ATP.
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• It contains vitamins because they are essential to make enzymes
work correctly. If enzymes do not work, then the cells are unable to
survive.
• It contains plant growth substances. These are critical for the
successful growth and differentiation of the callus because they
regulate growth and development. Two important plant growth
substances are auxins and cytokinins.
The following diagrams show the effect of different concentrations of
auxin and cytokinin on the differentiation of the callus:
(a)
Agar containing nutrients and
0.2 mgdm–3 cytokinin
The explant does not grow
(b)
Agar containing nutrients and
2mgdm–3 auxin
The cells of the explant grow
bigger
(c)
Agar containing nutrients and
2mgdm–3 auxin and 0.2 mgdm–3
cytokinin
A mass of undifferentiated cells
is produced
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(d)
Agar containing nutrients and
2mgdm –3 auxin and 0.02 mgdm–3
cytokinin
Roots form but shoots do not
(e)
Agar containing nutrients and
0.02mgdm –3 auxin and 1.0 mgdm–3
cytokinin
Shoots form but roots do not
These diagrams show that different concentrations of plant growth
substances greatly influence the growth of explants. The correct
concentrations are needed for shoot and root regrowth.
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IDENTIFICATION OF MICRO-ORGANISMS
SECTION 3
Identification of micro-organisms
One of the most fundamental tasks that someone working with microorganisms must perform is to identify micro-organisms. There are a
number of ways that this can be done. Firstly, the micro-organism can be
stained and viewed under the microscope. This gives information
regarding the shape (morphology) of the micro-organism. Another way
of identifying micro-organisms is to find out the reactions that the
micro-organism carries out. This is known as biochemical testing.
Once the morphology and biochemistry of the micro-organism is
known, identification keys and tables can be consulted to work out the
identity of the micro-organism being investigated.
Use of the microscope in identifying micro-organisms
Micro-organisms are often difficult to observe using a microscope and so
they are generally stained before being viewed.
A stain that is commonly used in the initial identification of a bacterium
is the Gram stain. It identifies bacteria as being gram positive or gram
negative, depending on the type of cell wall present in the bacterium.
Gram-positive bacteria appear purple when viewed under the
microscope whereas gram-negative bacteria appear red.
(The gram stain is also discussed in Section 1 of the Student Materials
for Unit 1: Microbiology – see page 8.)
When viewed under a microscope, bacteria are observed to have a
definite shape (morphology). Three shapes are commonly seen –
round, rods and spirals.
Figure 11
Round bacteria are called cocci:
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IDENTIFICATION OF MICRO-ORGANISMS
Rod-shaped bacteria are called bacilli:
Spiral bacteria are called spirilla:
Special stains are used to show the presence of structures in a
bacterium. As mentioned, the gram stain reveals the type of cell wall
present. Other stains are used to show the presence of capsules and
flagella. A capsule is a protective layer surrounding some bacterial cells.
Flagella are filaments used by bacteria for movement. The presence,
number and arrangement of flagella are used to identify bacteria. Some
bacteria have no flagella, others have a single flagellum at one end of the
cell, while others have flagella surrounding the cell.
Fungi can be identified under the microscope by observing the shape
and arrangement of their spore-bearing structures (sporangia).
(The structure of micro-organisms is discussed in Section 1 of the
student materials for Unit 1: Microbiology)
Biochemical tests
These tests give information about the reactions that a micro-organism
carries out, such as whether it requires oxygen for growth or whether it
ferments a particular sugar to produce acid. Each species of bacterium
has a characteristic profile or ‘fingerprint’ of biochemical tests that can
be used to identify it.
Some of the tests that are carried out are described in the following
paragraphs.
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Extracellular digestion
In these tests, micro-organisms are inoculated on different types of
medium. If the micro-organism produces extracellular enzymes, then it
is able to grow on the medium. Examples include growing microorganisms on media containing starch, casein, gelatine and fat.
For example, if a micro-organism is inoculated onto agar medium
containing starch and if it produces amylase (which digests starch), then
the micro-organism grows on this medium. The digestion of the starch
can be observed by staining the agar plate with iodine.
Fermentation of carbohydrate
Micro-organisms are grown in media containing different carbohydrates
and the production of acid or acid and gas is recorded. For example,
the following table shows the characteristic results of two different
bacteria when grown in the presence of glucose and lactose:
Bacteria
Lactose fermentation
Acid
Gas
produced
produced
Glucose fermentation
Acid
Gas
produced
produced
E.coli
+
+
+
+
Shigella
–
–
+
–
(+) means that acid/gas is produced
(–) means that acid/gas is not produced.
Catalase test
A sample of viable bacteria is mixed with a solution of hydrogen
peroxide. If catalase is present, oxygen is produced and frothing of the
sample is observed.
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IDENTIFICATION OF MICRO-ORGANISMS
Oxidase test
Cytochrome c is used to test for the presence of the enzyme oxidase.
Flowcharts are used to identify bacteria based on their morphology and
biochemical tests. An example of a flow chart is shown:
Figure 12
From this chart, a bacterium that is gram positive, catalase negative and
found in a chain arrangement can be identified as being Streptococcus.
You have now completed the theory content for Unit 2:
Microbiological Techniques, and you should be able to put this
theory into practice when you work with micro-organisms in the
laboratory.
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BIBLIOGRAPHY
Some suggested reading materials for teachers/lecturers
The following is a commentary on some published reading materials that
may be useful when delivering Higher Biotechnology. This list is in no
way exhaustive and is meant only as a starting point for any tutor
delivering the units for Higher Biotechnology for the first time.
Foundations in Microbiology (3rd edition)
by Kathleen Park Talaro and Arthur Talaro
Published by WCB/McGraw-Hill
ISBN: 0-697-35452-0
This is a general introductory microbiology book that is a good teacher’s
resource, especially if you do not have a microbiology background. The
book is aimed at undergraduates, so it is too detailed and advanced to
be used as a student resource. But it is easy to read and has lots of good
illustrations and diagrams. There is an interactive CD-ROM that can be
purchased to accompany the book. It provides lots of detailed
background knowledge on many of the topics in all of the three units
that comprise Higher Biotechnology.
Fundamentals of Microbiology (5th edition)
by I Edward Alcamo
Published by Benjamin/Cummings Publishing Company
ISBN: 0-8053-0532-7
This is another general microbiology book that is a good teacher’s
resource. Again, it is easy to read with lots of diagrams and anecdotes
(although they are all American). This book is a good source of graphs
that could be the basis for problem-solving questions. It also provides
lots of detailed background information for all three units of Higher
Biotechnology.
Micro-organisms and Biotechnology (1st and 2nd editions)
by Jane Taylor
Published by Nelson Thornes
ISBN: 0-17-448255-8 (second edition)
This book is now into its second edition and may be used as a teacher
and student resource. Both the first and second edition provide
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BIBLIOGRAPHY
background knowledge for all three units comprising Higher
Biotechnology and the book is especially good for the enumerating
micro-organisms section in Unit 2 (Microbiological Techniques). The
second edition also covers some ethical issues surrounding some
biotechnology processes.
Basic Biotechnology (2nd edition)
Edited by Colin Ratledge and Bjorn Kristiansen
Published by Cambridge University Press
ISBN: 0-521-77917-0
This is a book for teachers who are enthusiasts and want to have a
detailed knowledge of biotechnology. It provides all the background
knowledge (and more!) required for delivering Unit 3 (Biotechnology).
Some suggested websites
www.Biotechinstitute.org
This is an American website that has lots of biotechnology information. It
has links to biotechnology-related news stories from a range of sources,
e.g. ‘Nature’, Yahoo and the BBC. There are teachers’ resources and
links to other websites. Also, you can download back copies of the
magazine Your World; this is aimed at post-16 students. Each issue
covers one particular biotechnology topic and so can be used as a
classroom resource.
www.biowise.org.uk
This website provides downloadable case studies on industrial
biotechnology that may be useful for Unit 3 (Biotechnology). The case
studies highlight companies in the UK that actively use biotechnology;
so they are a good introduction to students to show the practical
relevance of what they are studying.
www.sgm.ac.uk
This is the Society for General Microbiology website which has links to
current ‘hot’ topics and news items, so it is a good way of keeping up to
date with issues in microbiology. It also has educational resources and
links to online microbiology resources.
www.ncbe.reading.ac.uk
This website provides downloadable protocols for practical exercises, as
well as online learning materials. It has a good section on safety issues to
be taken into consideration when carrying out biotechnology practical
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exercises. It also provides information about the Scottish Centre for
Biotechnology Education.
www-saps.plantsci.cam.ac.uk
This website has protocol information, details on how to purchase kits
that can be used as learning activities, and details of biotechnology
workshops for teachers and the annual biotechnology summer school.
www.scottishbiotech.org
This is the website of the Scottish Colleges Biotechnology Consortium
who deliver technical training to industry and schools. Online courses
are available.
www.sserc.org.uk
This website provides information about the Scottish Institute of
Biotechnology Education (SIBE), which runs workshops for teachers
and pupils. Members can access an interactive manual on Microbiological
Techniques for schools and colleges. It includes a code of practice on
Safety in Microbiology and notes on Micro-organisms for Investigations.
www.sebiotech.org.uk
This is the website of Scottish Enterprise that is dedicated to the
Scottish biotechnology industry. It is very useful for keeping up to date
with the activities of biotechnology companies in Scotland.
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ADVICE FOR OUTCOME 2
APPENDIX
Advice for Outcome 2 for teachers/lecturers
Outcome 2 is a practical-based outcome and candidates are required to
carry out and be familiar with techniques relating to growth limitation
and sterilisation, culturing micro-organisms and identifying microorganisms. This outcome is designed to put the knowledge and
understanding from Outcome 1 into practice.
There are two very good resources that are available that provide the
majority of protocols needed to successfully deliver this practical
outcome.
HSDU Intermediate 2 Biotechnology support materials for the unit
‘Working with Micro-organisms’ provide detailed, illustrated protocols
for:
•
•
•
•
•
•
General aseptic technique
Pouring plates
Subculturing micro-organisms
Isolating pure cultures
Use of the microscope
Staining of micro-organisms (but not Gram stain).
These support materials are available for students and there are also
separate materials for lecturers/technicians.
HSDU/SSERC Biology/Biotechnology Microbiology Techniques
(Intermediate 1 – Advanced Higher) support materials is a step-by-step
manual that provides a number of detailed protocols for techniques
required in this unit, such as:
•
•
•
•
•
Aseptic technique
Media preparation
Subculturing
Staining
Enumerating micro-organisms.
Used together, both of these resources provide sufficient information
for successfully delivering the practical component of this unit.
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