NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Bacterial Transformation (pGLO)
Support Materials
[HIGHER]
The Scottish Qualifications Authority regularly reviews
the arrangements for National Qualifications. Users of
all NQ support materials, whether published by
Learning and Teaching Scotland or others, are
reminded that it is their responsibility to check that the
support materials correspond to the requirements of the
current arrangements.
Acknowledgement
Learning and Teaching Scotland gratefully acknowledges this contribution to the National
Qualifications support programme for Biology.
The protocols contained within this booklet are amalgamated from various sources including
SSERC, LT Scotland NQ Curriculum Support, HSDU, SAPS, Bio-Rad and NCBE. Each
protocol is acknowledged to the appropriate source.
Power point taken from Biotechnology PowerPoint Presentation Unit 1-3
Unit 1c Genetic Engineering
Every effort has been made to trace all the copyright holders but if any have been inadvertently
overlooked, the publishers will be pleased to make the necessary arrangements at the first
opportunity.
© Learning and Teaching Scotland 2011
This resource may be reproduced in whole or in part for educational purposes by educational
establishments in Scotland provided that no profit accrues at any stage.
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© Learning and Teaching Scotland 2011
Contents
Introduction
4
Teacher support notes
5
Teacher support notes: Bio-Rad pGLO activity
6
Student support notes: Bio-Rad pGLO activity
8
Teacher support notes: NCBE activity
10
Student support notes: NCBE activity
12
Student revision questions
13
Student brief: research activity
14
Extra guidance on research task
15
Scientific communication activity
16
Scientific communication example 1: scientific poster
17
Scientific communication example 2: PowerPoint
18
Health and safety: Bio-Rad pGLO activity
19
Health and safety: NCBE activity
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Introduction
Higher Biology to support: Unit 2 – Metabolism and Survival part 3: The
control of metabolism in microorganisms, Recombinant DNA technology
Resources
A PowerPoint on the background of transformation and cloning is supplied
with this pack. Below is a slide from the PowerPoint.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Teacher support notes
Activities
Two practical activities are explained, one using a pGLO bacterial
transformation kit and one using an NCBE transformation kit.
Teachers may wish to take into account the number of students doing this
activity when they choose between these two practical activities – see pages 6
and 10 of this document for cost and numbers of students that can participate.
Bacterial transformation
Recombinant DNA technology produces recombinant DNA. Recombinant
DNA is a form of engineered DNA that is produced by combining two or
more sequences of DNA. To carry out recombinant DNA technology a gene
of interest is isolated and then inserted int o a vector. A vector acts as a
molecular vehicle to integrate foreign DNA into a host’s genome . One such
example of a vector is a bacterial plasmid, which contains extrachromosomal
circular DNA molecules that can be transferred into a bacteria genome ; this
process is known as bacterial transformation. The gene of interest , together
with the DNA that comprises the plasmid, are referred to as recombinant
DNA. The host cell expresses the foreign protein from the recombinant DNA.
Recombinant DNA technology is used in many exciting areas of
biotechnology. For example, in agriculture, genes can be introduced which
increase yield. In medicine, insulin can be produced to treat diabetes. In
laboratory work genes can be introduced to microorganisms which render
them harmless in an external environment.
 The PowerPoint (Bacterial Transformation) supplied with this pack
provides useful background information for students.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Teacher support notes: Bio-Rad pGLO activity
Cost




Cost is based on 32 students working in group s of 4.
pGLO bacterial transformation kit costs £65.
pGLO bacterial transformation refill package costs £38.
pGLO plasmid only costs £19.
 The above can all be purchased via the Bio-Rad website.
The first year that this practical activity is carried out th e full kit would be
required by most schools, but it is worthwhile checking the kit contents on
the website. If your school does work with microorganisms you may be able
to purchase the refill or plasmid only. For a list of all the extra equipment
needed that is not included in the kit see the Technician’s Guide in this pack.
Duration – three or four lessons
In this practical activity students will
transform Escherichia coli bacteria with the
gene that encodes a green fluorescent protein
(GFP). In other words they will insert a
plasmid that contains the gene for GFP into
bacteria. GFP is a protein produced by the
bioluminescent jellyfish Aequorea victoria.
GFP causes the jellyfish to glow under
ultraviolet (UV) light. The plasmid used in
this activity is called pGLO. The pGLO
plasmid contains a gene for the GFP and a
gene for resistance to the antibiotic
amplicillin. The GFP gene can be turned on
in transformed cells by adding the sugar
arabinose to the cells’ nutrient medium.
Without arabinose the bacteria will not fluoresce and will appear white. With
arabinose the bacteria will fluoresce green under a UV light. The nutrient
medium that the bacteria are grown on contains ampicillin. The ampicillin
kills all bacteria that do not contain the plasmid because they do not have
resistance, therefore if any bacteria grow on the plate they must have been
transformed (contain the pGLO plasmid with the GFP gene and the resistance
to ampicillin gene). The E. coli bacteria now exhibit two new traits: they are
able to grow on ampicillin and they glow green.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
In this activity students will learn about the process of moving genes from
one organism to another with the aid of a plasmid and get to practise
techniques used in microbiology.
 Bacteria will fluoresce green.
 Bio-Rad provide a pGLO manual that you are able to download free of
charge via their website (www.bio-rad.com). Search for ‘pGLO bacterial
transformation kit’ in the search box then select the link for this. Scroll
down the page until you find the download PDF of the kit curriculum.
 This document contains a detailed instructor’s guide, including points to
emphasise to help the experiment run smoothly, and a student manual. The
student manual provides four detailed les sons (see below).
 The lessons are designed to encourage students to think about the
procedures they are carrying out and analyse the data from their results.
 The lessons provide the background information the students require along
with questions, experiment instructions, an opportunity for students to
analyse their results and an extension activity that allows them to evaluate
the effectiveness of the procedure.
Lesson structure
Lesson 1
Introduction.
Lessons 2 and 3
The practical work requires two ~50-minute lessons with an overnight
incubation in between.
Lesson 4 (extension)
Calculating the transformation efficiency.
Extra information
Preparation time
You need to start preparing 3 to 7 days in advance to allow time to set up the
agar plates (3 days if you have an incubator, 7 if you do not).
Points to note
Although the pGLO manual gives clear instructions on how to do the
practical techniques for this experiment some students may still find some of
the practical work challenging. To overcome t his it may be useful to practise
some of the procedures beforehand with your students, e.g. using pipettes and
loops practise streaking and spreading the bacteria.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Student support notes: Biorad pGLO activity
In this activity you are going to transform E. coli bacteria with the gene that
codes for green fluorescent protein (GFP). In other words you are going to
insert a plasmid that contains the gene for GFP into bacteria. GFP is a
protein produced by the bioluminescent jellyfish Aequorea victoria. GFP
causes the jellyfish to fluoresce and glow under UV light.
The plasmid used in this activity is called pGLO. The pGLO plasmid
contains a gene for the GFP and a gene for resistance to the antibiotic
amplicillin. The GFP gene can be turned on in transformed c ells by adding
the sugar arabinose to the cells’ nutrient medium. Without arabinose the
bacteria will not fluoresce and will appear white. With arabinose the bacteria
will fluoresce green.
The nutrient medium that the bacteria are grown on contains ampici llin. The
ampicillin kills all bacteria that do not contain the plasmid because they do
not have resistance, therefore if any bacteria grow on the plate they must
have been transformed (contain the pGLO plasmid with the GFP gene and
the resistance to ampicillin gene). The E. coli bacteria now exhibit two new
traits: they are able to grow on ampicillin and they glow green.
In this activity, you will learn about the process of moving genes from one
organism to another with the aid of a plasmid and get to pra ctise techniques
used in microbiology.
Problem
Can the bacteria E. coli be transformed by plasmid DNA to enable us to
observe the acquired phenotypic trait of GFP exhibited by the transformed
bacteria cells?
Hypothesis
The transformed E. coli cells will fluoresce green under a UV light source.
Procedure
Your teacher will give you a student manual that will help to instruct you
through this practical activity.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Scientific communication
You should look to present your work in a suitable way, for exam ple in the
form of a laboratory report, scientific poster or presentation. Suitable
examples are provided on pages 17 and 18.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Teacher support notes: NCBE activity
Cost
 Cost is based on 16 students working in groups of 2.
 Bacterial transformation kit
 Replacement parts (as provided in the kit)
Slope culture of E. coli K12 STRAIN DH5α18
p2K plasmid DNA
Kanamycin/X-Gal/LB agar + LB agar tablets
Transformer kit Student’s guides, 8
All the above can be purchased via www.ncbe.reading.ac.uk/
£70
£10
£12
£16
£12
The first year that this practical activity is carried out the fu ll kit would be
required by most schools, but it is worthwhile checking the kit contents on
the website. If your school does work with microorganisms you may be able
to purchase replacements parts only.
NCBE recommend that students do not work in groups o f more than two for
this activity:
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/PDF/NCBEpricelist.pdf
Duration – three lessons
In this activity students will learn about the structure of bacteria and bacterial
transformation by carrying out basic practical techniques used in
microbiology. They are also encouraged to think about the ethical, social and
safety issues involved in bacterial transformation.
The host strain of E. coli used in this activity cannot hydrolyse the sugar
lactose because it lacks the gene for the enzyme β-galactosidase that does
this. However, the plasmid used does carry this gene. If the host bacterium
takes up the plasmid it can hydrolyse lactose. A colourless compound called
X-Gal is added to the agar plate. X-Gal can also be hydrolysed by βgalactosidase, yielding galactose and an insoluble blue dye. The dye is
precipitated within the bacteria, enabling X -Gal to be used as an indicator of
β-galactosidase activity. The antibiotic kanamycin is also added to the agar
plates. The transformed cells have partial resistance to this antibiotic while
the cells without the plasmid do not survive, therefore the bacterial cells that
survive have been transformed and can be easily identified because they are
blue.
 Student, Teacher and Technician’s guides are availa ble to download on the
following website:
http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/transformer.html
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
 The Student’s guide gives detailed background information about DNA
and bacterial transformation. It then provides instructions for the practical
technique and examples of further investigations that could be carried out.
Lesson structure
Lesson 1
Introduction and background.
Lesson 2
Practical work.
Lesson 3
Results (plus possible extension – calculating the transformation efficiency).
Extra information
Preparation time
The stock plates of bacterial cultures should be prepared 3 to 4 days in
advance. All other preparation should be done when convenient (no more than
7 days in advance) or immediately before the practical lesson.
A detailed timeline for this is laid out in page 7 of the Technician’s guide.
Page 8 in the Technician’s guide gives a list of the apparatus that each
student should have at their workbench.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Student support notes: NCBE activity
In this activity you will learn about the structure of bacteria and bacterial
transformation by carrying out basic practical techniques used in
microbiology. You will also be encouraged to think about the ethical, social
and safety issues involved in bacterial transformation.
The host strain of E. coli used in this activity cannot hydrolyse (break down
by chemical reaction using water) the sugar lactose because it lacks the gene
for the enzyme β-galactosidase that does this. However, the plasmid you are
using does carry this gene. If the host bacterium takes up the plasmid it can
now hydrolyse lactose. The agar plates that the bacteria are grown on contain
a colourless compound called X-Gal. X-Gal can also be hydrolysed by βgalactosidase, producing galactose and an insoluble blue dye. The dye is
precipitated within the bacteria, enabling X -Gal to be used as an indicator of
β-galactosidase activity. The antibiotic kanamycin is also added to the agar
plates. The transformed cells have partial resistance to this antibiotic, while
the cells without the plasmid do not survive, therefore the bacterial cells
which survive have been transformed and can be easily identified because
they are blue.
Problem
Can the bacteria E. coli be transformed by a plasmid DNA to enable the
bacteria to produce the enzyme β-galactosidase?
Hypothesis
The transformed E. coli cells will be visible as blue colonies because the β galactosidase will hydrolyse X-Gal into galactose and an insoluble blue dye.
Procedure
Your teacher will give you a student manual that will help to instruct you
through this practical activity.
Scientific communication
You should present your work in a suitable way, for example in the form of a
laboratory report, scientific poster or presentation. Suitable examples are
provided on pages 17 and 18.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Student revision questions
1.
What is rDNA?
2.
What is rDNA technology used for?
3.
One way of producing rDNA is by bacterial
transformation.
What is bacterial transformation?
4.
What is a vector?
5.
Name the type of vector used in bacterial transformation.
6.
Put the following bacterial transformation steps in the correct order.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
A restriction enzyme is used to extract the gene of interest.
The gene and the plasmid now have complimentary sticky ends so
the gene can be inserted into the plasmid.
The bacteria reproduce and each new bacterium has the desired
gene and can therefore produce the desired protein.
DNA ligase is used to seal the fragments.
The gene of interest is isolated.
The same restriction enzyme is used to cut the plasmid.
The plasmid is now inserted into bacteria.
7.
During bacterial transformation the gene for resistance to an antibiotic,
for example ampicillin, is usually inserted into the plasmid along with
the gene of interest. Explain why.
8.
Recombinant DNA technology has many exciting current appli cations.
Pick one of these and give a detailed account of the processes involved
and the product.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Student brief: research activity
A breakthrough in recombinant DNA technology occurred in 1977 when
Herbert Boyer produced biosynthetic human insulin in the laboratory.
Recombinant DNA technology has many future applications that are being
researched in laboratories now. These technologies have raised important
ethical questions.
In carrying out your research you should answer the following questions:
 What are the main current applications of recombinant DNA technology?
 What are the future applications of recombinant DNA technology?
 Why is recombinant DNA technology important to the world’s future?
 Why is it important to consider the ethical and economic implications of
recombinant DNA technology ?
Answer the questions by carrying out research, which will probably be web based. You are advised to have completed the Web Research in Biology
activity on the LTS website before attempting this activity.
You may work individually or as part of a team.
Produce a report of your findings. This may be hand written, printed or
electronic and saved in an e-portfolio.
You should spend approximately 2 hours on this activity.
If you work as part of a team that produces one report, you should include a
short statement at the end of the report that indicates which part of the work
you were responsible for.
There are some more pointers to help you with your research on the next
page.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Extra guidance on research task
Things to consider when undertaking web-based research
Stay focused!
Make sure that you remain focused as you carry out your research. It is very
easy to get side-tracked. Make sure you have clear questions that you wish to
answer. Keep reminding yourself what you are trying to find out as you surf.
Interesting, but not relevant, sites can be re -visited later.
Don’t write your report as you go along
Your task is to find suitable sites that may contain the information you
require. Sites that seem to be promising can be bookmarked so that they can
be returned to later. Tables, graphs and pictures can be copied into a folder. It
is likely that some will be used and some will not. It is important to make a
selection later, not just as you stumble across the site.
It may help to include an introduction at the start of the report and it is often
best if this is written last. The introduction can act as a summary of the
findings and is best written after the information has been collected.
Team work
If working as part of a team, it is important to allocate specific tasks so that
each individual does not waste time answering the same question. However,
if a piece of information is particularly hard to find, it may help to have a
combined search for that particular item. Liaison between team members is
particularly important.
If you are working as part of a team, and you have produced a single report
between you, you should include a short statement at the end of the report
that indicates which part of the work you were responsible for.
Cut and paste - DON’T
The finished report should be easy to read. Avoid simply cutting and pasting
large chunks of text. Try to summarise the information you find and where
possible use your own words to state what you have found . Tables and charts
can be very useful but often contain too much data. If information is provided
in a table, consider extracting the data you require and making your own table
and graph.
Referencing
It is important that the information you include in yo ur report is referenced.
As a general rule, someone else should be able to easily find where you got
your information from. Make sure that your report includes a clear indication
of where you have sourced your data.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Scientific communication activity
The aim of this activity is to compare a number of different ways of
presenting investigation results in a scientific communication.
Two scientific communications are presented in this pack. They include:
1.
2.
a PowerPoint presentation (thumbnails of each page)
a scientific poster (the original is A3 – this pack includes an A4
version).
Study each of the two formats above and a basic laboratory report, and
consider the following questions:
1.
Which of the formats contains the most detail?
2.
Which format is most visually appealing?
3.
Which format is most likely to be seen/read by the largest audience?
4.
Which format is easiest for an individual/group to produce?
Remember that for assessment purposes you need to produce a scientific
communication that has the following:




a clearly identified aim
an analysis of the results
valid conclusions
an evaluation of experimental procedures (if relevant).
Other formats suitable for presenting scientific information include the
following:
 magazine or newspaper article
 web page
 video.
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Scientific communication example 1: scientific poster
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© Learning and Teaching Scotland 2011
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Scientific communication example 2: PowerPoint
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© Learning and Teaching Scotland 2011
CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Health and safety: Biorad pGLO activity
The work in this document is classed as level 3 safety. The recommendation
from SSERC (http://www.science3-18.org/) regarding this is that the school
should have one person in the department trained to level 3. This is usually
the technician as they will be doing the preparation and disposal of plates,
organisms etc.
SSERC have also noted that the organisms must be destroyed by autoclaving
within 1 week of the end of the practical.
The E. coli bacteria HB101 K-12 strain contained in this kit is not a
pathogenic organism.
Handling of the E. coli K-12 strain requires the use of standard
microbiological practices.
These practices include, but are not limited to, the following:
Preparing work surfaces
Freshly diluted 1% hypochlorite solutions (a good quality commercial bleach,
eg Domestos, Chloros or laboratory sodium chlorate(I) [hypochlorite]) may
also be used to disinfect non-absorbent surfaces but care must then be taken
to avoid contamination of the skin or clothes. It is good practice to use a
different disinfectant for swabbing work surfaces.
A spillage kit
 A screw-capped bottle containing a measured quantity of undiluted
disinfectant with the correct volume of water needed to dilute it marked on
the side.
 A quantity of paper towels.
 A pair of autoclavable tongs or a small plastic dustpan.
 A pair of disposable plastic gloves.
 An autoclavable waste disposal bag.
 An autoclavable container, with suitable means of closure into which
contaminated, broken glass may be easily transferred for sterilisation.
Everyone working with microorganismsms must:
 Wash their hands thoroughly, using soap and water, both before and after
microbiology work
 Cover exposed cuts with waterproof dressings.
 Tie long hair back.
 Perform all procedures carefully to minimise the creation of aerosols .
 Use mechanical pipeting devices.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
 Not eat, drink, smoke or apply cosmetics in the work area.
 Wear protective eyewear and gloves.
Use an autoclave if possible. If an autoclave is not available, all solutions and
components (loops and pipettes) that have come into contact with bacteria
can be placed in a fresh 10% bleach solution for at least 20 minutes for
sterilisation. A shallow pan of this solution should be placed at every
laboratory station. No matter what you choose, all used loops and pipettes
should be collected for sterilisation.
Petri dishes should normally be of the disposable plastic type. If glass Petri
dishes are used they must be sterilised for re -use by autoclaving or with dry
heat. Where glass Petri dishes are re-used chemical disinfection is not
acceptable for the purposes of sterilisation.
Safety glasses are recommended when using bleach solutions.
Ampicillin may cause allergic reactions or irritation to the eyes, respiratory
system and skin. In case of contact with eyes, rinse immediately with plenty
of water and seek medical advice. Ampicillin is a member of the penicillin
family of antibiotics. Those with allergies to penicillin or to any other
member of the penicillin family of antibiotics should avoid contact with
ampicillin.
Ultraviolet lamps
UV radiation can cause damage to eyes and skin. Shortwave UV light is more
damaging than long-wave UV light. The Bio-Rad UV lamp recommended is
long wave.
If possible, use UV-rated safety glasses or goggles.
References
Biology/Biotechnology Safety in Microbiology, A Code of Practice for
Scottish Schools and Colleges, SSERC Limited
Biotechnology Explorer, pGLO™ Bacterial Transformation Kit
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Health and Safety: NCBE activity
Health and safety instructions are given in the NCBE Teacher’s guide.
Extra information gathered from SSERC is given below.
This work is classed as level 3 safety. The recommendation from SSERC
regarding this is that the school has one person in the department trained to
level 3. This is usually the technician as they will be doing the preparation
and disposal of plates, organisms etc.
SSERC have also noted that the organisms must be destroyed within 1 week
of the end of the practical by autoclaving.
The E. coli bacteria K-12 DH5α18 strain contained in this kit is not a
pathogenic organism.
Handling of the E. coli K-12 strain requires the use of standard
microbiological practices.
These practices include, but are not limited to, the following:
Preparing work surfaces
Freshly diluted 1% hypochlorite solutions (a good quality commercial bleach,
eg Domestos, Chloros or laboratory sodium chlorate(I) [hypochlorite]) may
also be used to disinfect non-absorbent surfaces but care must then be taken
to avoid contamination of the skin or clothes. It is good practice to use a
different disinfectant for swabbing work surfaces.
A spillage kit
 A screw-capped bottle containing a measured quantity of undiluted
disinfectant with the correct volume of water needed to dilute it marked on
the side.
 A quantity of paper towels.
 A pair of autoclavable tongs or a small plastic dustpan.
 A pair of disposable plastic gloves.
 An autoclavable waste disposal bag.
 An autoclavable container, with suitable means of closure into which
contaminated, broken glass may be easily transferred for sterilisation.
During practical work:
 Work should be carried out close to a Bunsen burner flame.
 If wire loops are used these should be heated with a blue flame until they
are red hot.
BACTERIAL TRANSFORMATION (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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CASE STUDY: BACTERIAL TRANSFORMATION (pGLO)
Everyone working with microorganismsms must:
 Wash their hands thoroughly, using soap and water, both before and after
microbiology work
 Cover exposed cuts with waterproof dressings.
 Tie long hair back.
 Perform all procedures carefully to minimise the creation of aerosols.
 Use mechanical pipeting devices.
 Not eat, drink, smoke or apply cosmetics in the work area.
 Wear protective eyewear and gloves.
Use an autoclave if possible. If an autoclave is not available, all solutions and
components (loops and pipettes) that have come in contact wit h bacteria can
be placed in a fresh 10% bleach solution for at least 20 minutes for
sterilisation. A shallow pan of this solution should be placed at every
laboratory station. No matter what you choose, all used loops and pipettes
should be collected for sterilisation.
Petri dishes should normally be of the disposable plastic type. If glass Petri
dishes are used they must be sterilised for re -use by autoclaving or with dry
heat. Where glass Petri dishes are re-used chemical disinfection is not
acceptable for the purposes of sterilisation.
Safety glasses are recommended when using bleach solutions.
References
Biology/Biotechnology Safety in Microbiology, A Code of Practice for
Scottish Schools and Colleges, SSERC Limited.
NCBE Teacher’s Guide.
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