NATIONAL QUALIFICATIONS CURRICULUM SUPPORT
Biology
Unit 1
Activities
[REVISED ADVANCED HIGHER]
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Acknowledgements
The publisher gratefully acknowledges permission to use the following sources: image of haemoglobin
from http://commons.wikimedia.org.uk/wiki/File:1GZX_Haemoglobin.png and image of a nucleosome
from http://commons.wikimedia.org/wiki/File:Nucleosome_structure.png both © Richard Wheeler
(Zephyris); image of beta sheets from http://commons.wikimedia.org/wiki/File:PDB_1jy6_EBI.jpg ©
http://www.ebi.ac.uk; image of kinases from http://commons.wikimedia.org/wiki/File:Ch4_kinases.jpg ©
National Institute of General Medical Sciences; image of DNA X-ray from
http://commons.wikimedia.org/wiki/File:ABDNAxrgpj.jpg, ‘Physical Chemistry of Food’, vol. 2, van
Nostrand Reinhold: New York, 1994, I.C. Baianu et al; image of a protein primary structure from
http://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg and image of DNA Exons from
http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif both © The National Human Genome
Research Institute; image of electrophoresis from http://commons.wikimedia.org/wiki/File:SDSPAGE_Electrophoresis.png © Bensaccount at en.wikipedia; image no 3418 of African sleeping sickness
from http://phil.cdc.gov/phil/details.asp © CDC/Alexander J. da Silva, PhD/Melanie Moser; image no
11820 of Giemsa-stained light photomicrograph revealed the presence of a Trypanosoma brucei parasite,
which was found in a blood smear from http://phil.cdc.gov/phil/details.asp © CDC/Blaine Mathison;
image from Toxicology in Vitro 18 (2004) 1–12, Workshop report, The humane collection of fetal bovine
serum and possibilities for serum-free cell and tissue culture, reprinted from Toxicology in Vitro 18, Vol
1-12, Workshop report, The humane collection of fetal bovine serum and possibilities for serum-free cell
and tissue culture by J. van der Valk,D. Mellor,R. Brands,R. Fischer,F. Gruber,G. Gstraunthaler,L.
Hellebrekers,J. Hyllner,F.H. Jonker,P. Prieto,M. Thalen,V. Baumans, 2004 with permission from Elsevier
http://www.journals.elsevier.com/toxicology-in-vitro/; image from article, Conservation, Variability and
the Modeling of Active Protein Kinases
http://www.plosone.org/article/slideshow.action?uri=info:doi/10.1371/journal.pone.0000982&imageURI
=info:doi/10.1371/journal.pone.0000982.g001 © 2007 Conservation, Variability and the Modeling of
Active Protein Kinases by James D. R. Knight, Bin Qian, David Baker, Rashmi Kothary; image from
article Proteomics of Trypanosoma evansi Infection in Rodents from
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.000979 © 2010 Proteomics of
Trypanosoma evansi Infection in Rodents by Nainita Roy, Rishi Kumar Nageshan, Rani Pallavi, Harshini
Chakravarthy, Syama Chandran, Rajender Kumar, Ashok Kumar Gupta, Raj Kumar Singh, Suresh
Chandra Yadav, Utpal Tatu; image of Signal transduction from
http://commons.wikimedia.org/wiki/File:Signal_transduction_v1.png © Roadnottaken at the English
language Wikipedia
© Crown copyright 2012. You may re-use this information (excluding logos) free of charge in any format
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2
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
Contents
Activity A: Health and safety
4
Activity B: Liquids and solutions
31
Activity C: Separation techniques
36
Activity D: Antibody techniques
45
Activity E: Microscopy
48
Activity F: Aseptic technique
51
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
3
ACTIVITY A
Activity A – Health and safety
Laboratory techniques for biologists: health and safety
Learning about risk assessments
Aim
In this activity you will become familiar with the purpose of making risk
assessments for practical activities in biology and practise carrying out and
writing up your own risk assessments.
Introduction
All the practical activities you have carried out in biology lessons will have been
risk assessed. If an activity involves the use of potentially hazardous substances
and/or procedures that carry a risk then a formal risk assessment will have been
carried out. A permanent written record of the risk assessment will be stored in
the department. Your teachers will have made themselves familiar with these
risk assessments to ensure that the activities are done safely.
What is a risk assessment?
A risk assessment is nothing more than a careful examination of what, in your
work, could cause harm to people, so that you can weigh up whether you have
taken enough precautions or should do more to prevent harm.
A risk assessment is not a familiar idea but you actually make them on a daily
basis whenever you do something that has the potential to cause harm, like
crossing a road or cooking a meal. In the workplace it is a legal requirement
that formal risk assessments are carried out.
The information needed to risk assess a laboratory -based activity is published
by a number of organisations, including the Consortium of Local Education
Authorities for the Provision of Science Services (CLEAPSS) and the
Scottish Schools Education Research Centre (SSERC). The Health and Safety
Executive (HSE) provides guidance on the Control of Substances Hazardous
to Health (COSHH) regulations. This information should be available as a
hard copy or electronically in your school’s science department.
4
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
A good source of information for writing risk assessments is the Student
Safety Sheets document published on the CLEAPSS website:
www.cleapss.org.uk/attachments/article/0/SSSA.pdf
Task 1: Learning about risk assessments for laboratory -based activities
To familiarise yourself with the features of risk assessments which are
relevant to your studies read:
 Section 96 of the CLEAPSS document at
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
 the information at http://www.bath.ac.uk/internal/biosci/bbsafe/assessments.htm
Task 2: Thinking about hazardous substances and the risk they pose
In laboratory-based activities you will often be working with hazardous
substances (substances that could cause you harm). List five hazardous
substances that you have worked with in biology lessons. For each substance
write down your ideas about the nature of its hazard and what you did in
order to minimise the likelihood that it would harm you (the risk). Compare
your ideas to the guidance given in the CLEAPSS document.
Task 3: Writing risk assessments for practical activities in biology
An example of a risk assessment for a practical activity (testing leaf sections
for starch) is given overleaf. The form’s layout is based on the standard
COSHH template. Read through the risk assessment and the methods for the
activity and check that the details match the guidance given in the CLEAPSS
document.
Now access the following three incomplete Risk Assessment Forms for the
following practical activities:
1.
2.
3.
DNA extraction
Food tests
Observation of cheek cells
Use the details given about the method for each activity and the guidance in
the CLEAPSS document to complete full risk assessments for each activity.
Ask your teacher to check and sign off your completed risk assessments.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
5
ACTIVITY A
Example risk assessment form
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION1: EVALUATING THE RISKS
1A Title of
activity
1B Brief
summary of
procedure
being risk
assessed
Testing pieces of leaves for starch
Pieces of leaves heated with boiling water and ethanol then stained with
iodine solution
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Ethanol
√
√
Iodine
(Low hazard
solution
if
concentration
< 1M)
1D Complete this section if microorganisms are being used
Genus and species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or
eyes
√
Ingestion
Inhalation
Skin absorption
√
√
√
1F Location of activity (tick all boxes applicable)
Open bench
√
6
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
Fume cupboard
Other (please specify)
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
protection
√
Face
protection
Hand
protection
Lab
coat
Respiratory
protection
Other (please
specify)
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
Ethanol discarded in organic waste jar and removed by technician immediately after
practical.
Pieces of leaves discarded in rubbish bin.
1I Spillage: State how spillages should be dealt with
For ethanol: shut off ignition sources.
For small volumes wipe up with cloth and rinse well.
For large spills cover with mineral absorbent (eg cat l itter) and scoop into bucket. If large
volume of ethanol spilt then open windows.
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
Eye: Wash with eyewash bottles/hose attached to tap/under gent ly-running water for 10
minutes – seek medical help.
Swallowing: Wash mouth with water and take small sips, do not induce vomiting – seek
medical help.
Skin contact: Wash with plenty of water. Remove contaminated clothing.
1K Other safety measures/considerations not already mentioned
Use water bath to heat small volume of ethanol. No naked flames.
Use small volume of dilute iodine solution (<1M)
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
7
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
Materials
Pieces of leaves, boiling tube, ethanol, white tile, iodine solution.
Method
Place pieces of leaves in a boiling tube with 5cm 3 water and heat for 2 minutes
at 90°C  Replace water with 5cm 3 ethanol and heat for 5 minutes at 90°C 
Remove leaf section and dip in small beaker of water  Spread leaf pieces on
white tile and add 2-3 drops of iodine solution
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
8
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Risk Assessment form: DNA Extraction
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION 1: EVALUATING THE RISKS
1A Title of activity
DNA extraction
1B Brief summary
of procedure being
risk assessed
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Ethanol
Protease
1D Complete this section if microorganisms are being used
Genus and Species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or
eyes
Ingestion
Inhalation
Skin absorption
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
9
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
protection
Face
protection
Hand
protection
Lab
coat
Respiratory
protection
Other (please
specify)
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
1I Spillage: State how spillages should be dealt with
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
1K Other safety measures/considerations not already mentioned
10
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
See attached sheet (next page)
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES/NO
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
11
ACTIVITY A
Extraction of DNA from fish eggs
Materials required per pair
15 ml of detergent @ 1: 10
dilution with distilled water
Ice-cold ethanol
1 heaped spatula of
lumpfish eggs
(‘caviar’)
3–4 drops of protease
enzyme
Test-tube and rack
3 spatulas of salt
Coffee filter and filter
funnel
Plastic dropping
pipette
Pestle and mortar
Method
1.
Add the fish eggs and salt to the mortar, then crush the eggs with the
pestle (the shells have to be broken and the proteins are precipitated by
the salt).
2.
Add the detergent to the mortar so that the liquid covers the fish eggs
completely (the detergent dissolves the lipids from the cell and nuclear
membranes).
3.
Add 3–4 drips of protease enzyme to the mixture and stir vigorously
(the protease will partially degrade any soluble proteins).
4.
Filter the mixture through the filter (check it for holes first) and collect
the filtrate in the test-tube.
5.
Set your test-tube at an angle in the test-tube rack. Add the ice-cold
ethanol by very carefully pouring it down the side of the tube (slowly
and gently). DNA precipitates as long threads in cold ethanol and can
be found at the interface between the detergent solution and the ethanol.
6.
You could try to pick up some of the DNA by gently winding a mounted
needle plastic hook around in the DNA and lifting it up.
12
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Risk assessment form: Food Tests
SECTION 1: EVALUATING THE RISKS
1A Title of activity
Food tests
1B Brief summary
of procedure being
risk assessed
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Ethanol
Protease
1D Complete this section if microorganisms are being used
Genus and Species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or
eyes
Ingestion
Inhalation
Skin absorption
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
13
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
protection
Face
protection
Hand
protection
Lab
coat
Respiratory
protection
Other (please
specify)
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
1I Spillage: State how spillages should be dealt with
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
1K Other safety measures/considerations not already mentioned
14
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
See attached sheet (next page)
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES/NO
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
15
ACTIVITY A
Food testing activity
Materials and methods
Food samples to be tested: bread, ham, milk, green pepper, butter
The test for starch (the iodine solution test)
1.
2.
3.
Put a small sample of solid food on a spotting tile with a spatula OR
add 2 cm 3 of liquid food to a test-tube with a dropping pipette.
Add 2 drops of iodine solution from the bottle.
Observe the colour of the iodine solution where it touches the food.
The test for reducing sugars (the Benedict’s solution test)
1.
2.
3.
4.
5.
Mash up a small sample of food with a pestle and mortar.
Put the food in a test-tube with a spatula.
Add 2 cm 3 of Benedict’s solution from the bottle.
Heat the test-tube for 2 minutes in water at 90°C.
Remove the tube from the bath and observe the colour of the solution.
The test for protein (the Biuret solution test)
1.
2.
3.
4.
5.
Mash up a small sample of food with a pestle and mortar.
Put the food in a test-tube with a spatula.
Add 2 cm 3 of Biuret solution 1 to the test-tube and shake.
Add 3 drops of Biuret solution 2 to the test -tube.
Hold the test-tube up to the light and observe the colour of the solution.
The test for fat (the translucent spot test)
1.
2.
3.
Take a small sample of food and smear it on to the right-hand side of an
A4 piece of paper.
Put a small amount of water on the left -hand side of the paper.
Hold the paper up to the light and compare the two sides.
16
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Risk assessment form
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION1: EVALUATING THE RISKS
1A Title of activity
Observation of cheek cells
1B Brief summary
of procedure being
risk assessed
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
1D Complete this section if microorganisms are being used
Genus and species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or
Ingestion
Inhalation
Skin absorption
eyes
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
17
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
Face
Hand
Lab
Respiratory
Other (please
protection
protection
protection
coat
protection
specify)
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
1I Spillage: State how spillages should be dealt with
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
1K Other safety measures/considerations not already mentioned
18
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
Materials required
Sterile cotton bud, microscope slide, cover slip, bottle of methylene blue,
light microscope
Method
Swab inside of mouth  Smear cotton bud on small area of slide  Add 3
drops of methylene blue to stain cell sample  Cover with cover slip 
View with microscope
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES/NO
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
19
ACTIVITY A
Activity A: Risk Assessment Forms: Answers
Risk assessment form
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION1: EVALUATING THE RISKS
1A Title of activity
DNA extraction
1B Brief summary
of procedure being
risk assessed
Extraction of DNA from fish eggs using detergent, protease
enzyme and ethanol
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Ethanol
√
√
Protease
√
1D Complete this section if microorganisms are being used
Genus and species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or
Ingestion
Inhalation
Skin absorption
eyes
√
√
√
√
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
√
20
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
Face
Hand
Lab
Respiratory
Other (please
protection
protection
protection
coat
protection
specify)
√
1H Disposal of waste: State how hazardous material (including any microor ganisms) will
be disposed of safely
Extraction mixtures removed by technician immediately after practical.
1I Spillage: State how spillages should be dealt with
For ethanol: shut off ignition sources.
For small volumes wipe up with cloth and rin se well.
For large spills cover with mineral absorbent (eg cat litter) and scoop into bucket. If large
volume of ethanol spilt then open windows.
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
Eye: Wash with eyewash bottles/hose attached to tap/under gently -running water for 10
minutes – seek medical help.
Swallowing ethanol: Wash mouth with water and take small sips, do not induce vomiting
– seek medical help.
Swallowing protease: Dilute by drinking glass of water, do not induce vomiting – seek
medical help.
Skin contact with ethanol: Wash with plenty of water. Remove contaminated clothing and
rinse with water.
Skin contact with protease: Remove and rinse contaminated clothing. Wash skin with soap
and plenty of water.
1K Other safety measures/considerations not already mentioned
Stir mixture in mortar containing protease enzyme carefully.
Use volumes and concentrations of ethanol and protease that are as low as possible.
Enzymes may produce allergic reactions, consider use of gloves.
No naked flames.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
21
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
See attached sheet (next page)
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
22
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Extraction of DNA from fish eggs
Materials required per pair
15 ml of detergent @ 1: 10
dilution with distilled water
Ice-cold ethanol
1 heaped spatula of
lumpfish eggs
(‘caviar’)
3–4 drops of protease
enzyme
Test-tube and rack
3 spatulas of salt
Coffee filter and filter
funnel
Plastic dropping pipette
Pestle and mortar
Method
1.
Add the fish eggs and salt to the mortar, then crush the eggs with the
pestle (the shells have to be broken and the proteins are precipitated by
the salt).
2.
Add the detergent to the mortar so that the liquid covers the fish eggs
completely (the detergent dissolves the lipids from the cell and nuclear
membranes).
3.
Add 3–4 drips of protease enzyme to the mixture a nd stir vigorously
(the protease will partially degrade any soluble proteins).
4.
Filter the mixture through the filter (check it for holes first) and collect
the filtrate in the test-tube.
5.
Set your test-tube at an angle in the test-tube rack. Add the ice-cold
ethanol by very carefully pouring it down the side of the tube (slowly
and gently). DNA precipitates as long threads in cold ethanol and can
be found at the interface between the detergent solution and the ethanol.
6.
You could try to pick up some of the DNA by gently winding a mounted
needle/plastic hook around in the DNA and lifting it up.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
23
ACTIVITY A
Risk assessment form
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION1: EVALUATING THE RISKS
1A Title of activity
Food tests
1B Brief summary
of procedure being
risk assessed
Samples of food tested for starch, reducing sugars, proteins and
lipids using various reagents
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Iodine
(Low hazard
solution
for
concentrations
less than 1M)
Benedict’s
(Low hazard
solution
for
(copper (II)
concentrations
sulphate)
less than
0.5M)
Biuret
√
solution 1
(sodium
hydroxide)
Biuret
(Low hazard
solution 2
for
(copper (II)
concentrations
sulphate)
less than
0.5M
1D Complete this section if microorganisms are being used
Genus and species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
24
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin or Ingestion
Inhalation
Skin absorption
eyes
√
√
√
√
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
√
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
Face
Hand
Lab
Respiratory
Other (please
protection
protection
protection
coat
protection
specify)
√
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
Food/reagent mixtures removed by technicians.
1I Spillage: State how spillages should be dealt with
For small volumes wipe with damp cloth and rinse well.
For large spills cover with mineral absorbent (eg cat litter) and scoop into bucket.
Neutralise sodium hydroxide (alkali) with citric acid and rinse with water.
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
Eye: Wash with eyewash bottles/hose attached to tap/under running water for 10 minutes –
seek medical help.
Swallowing: Wash mouth with water and take small sips, do not induce vomiting – seek
medical help.
Skin: Wash with plenty of water. Remove contaminated clothing.
1K Other safety measures/considerations not already mentioned
Food is a biohazard. Food samples should not be tasted or consumed.
Be aware that some people are allergic to c ertain foods.
Use small volumes of reagents.
Benedict’s solution heated using water bath rather than naked flame.
Use dilute iodine solution and copper (II) sulphate solution (<1M).
Use dilute sodium hydroxide solution (<0.5M): it is not c orrosive at this concentration.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
25
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
See attached sheet (next page)
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………....................... Date: ……………
26
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
Food testing activity
Materials and methods
Food samples to be tested: bread, ham, milk, green pepper, butter
The test for starch (the iodine solution test)
1.
2.
3.
Put a small sample of solid food on a spotting tile with a spatula OR
add 2 cm 3 of liquid food to a test-tube with a dropping pipette.
Add 2 drops of iodine solution (this is brown) from the bottle.
Observe the colour of the iodine solution where it touches the food.
The test for reducing sugars (the Benedict’s solution te st)
1.
2.
3.
4.
5.
Mash up a small sample of food with a pestle and mortar.
Put the food in a test-tube with a spatula.
Add 2 cm 3 of Benedict’s solution (this is blue) from the bottle.
Heat the test-tube for 2 minutes in water at 90°C.
Remove the tube from the bath and observe the colour of the solution.
The test for protein (the Biuret solution test)
1.
2.
3.
4.
5.
Mash up a small sample of food with a pestle and mortar.
Put the food in a test-tube with a spatula.
dd 2 cm 3 of Biuret solution 1 (colourless) to the test-tube and shake.
Add 3 drops of Biuret solution 2 (faintly blue) to the test -tube.
Hold the test-tube up to the light and observe the colour of the solution.
The test for fat (the translucent spot test)
1.
2.
3.
Take a small sample of food and smear it onto the right -hand side of an
A4 piece of paper.
Put a small amount of water on the left -hand side of the paper.
Hold the paper up to the light and compare the two sides.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
27
ACTIVITY A
Risk assessment form
Complete this form before doing any activity/procedure involving risks to
health, including from a hazardous substance
SECTION1: EVALUATING THE RISKS
1A Title of
activity
1B Brief
summary of
procedure
being risk
assessed
Observation of cheek cells
Cell sample obtained from swabbing inside of mouth, stained with
methylene blue on slide and viewed using light microscope
1C Classification of named hazardous substance(s) used in the activity/procedure
Information in Griffin Education Catalogue, SSERC Hazardous Chemicals or
http://www.cleapss.org.uk/attachments/article/0/SSSPrint.pdf?...
Hazardous
Hazard (tick all boxes applicable)
substance
Corrosive
Dust
Flammable Harmful
Irritant
Toxic
Methylene
√
blue
Sodium
√ (5–10%
chlorate (I)
concentration)
1D Complete this section if microorganisms are being used
Genus and species
I have read the SSERC guidelines on
handling and safe disposal of
microorganisms (tick)
1E Route by which the substances are health hazards (tick all boxes applicable)
Direct contact: skin
Ingestion
Inhalation
Skin absorption
or eyes
√
√
√
√
1F Location of activity (tick all boxes applicable)
Open bench
Fume cupboard
Other (please specify)
√
28
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY A
1G Personal protective equipment requirements (tick all boxes applicable)
Eye
Face
Hand
Lab
Respiratory
Other (please
protection protection
protection
coat
protection
specify)
√
1H Disposal of waste: State how hazardous material (including any microorganisms) will
be disposed of safely
Used slides and swabs placed in beaker of disinfectant: sodium chlorate (I) (eg Milton or
Virkon) and removed by technician immediately after practical.
1I Spillage: State how spillages should be dealt with
For small volumes wipe with damp cloth and rinse well.
For large spills cover with mineral absorbent (eg cat litter) and scoop into bucket. If large
volume of disinfectant split then open windows.
1J Immediate remedial measures in the event of contamination: State how personal
injury will be minimised
Eye: Wash with eyewash bottles/hose attached to tap/under gently -running water for 10
minutes (20 minutes if disinfectant) – seek medical help.
Swallowing: Wash mouth with water and take small sips, do not induce vomiting – seek
medical help.
Skin: Wash with plenty of water (and soap if methylene blue). Remove contaminated
clothing and rinse with water.
1K Other safety measures/considerations not already mentioned
Cheek cells are a biohazard (tiny risk of transmission of HIV/hepatitis virus). Use sterile
cotton buds for single individual use only and immediately place in disinfectant after use.
Learners should only handle samples from their own body.
Use low volume and concentration of methylene blue dye.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
29
ACTIVITY A
Section 2: detailed description of the activity/procedure (alternatively the
appropriate instruction sheet can be stapled to this form)
Materials required per individual
Sterile cotton bud, microscope slide, cover slip, bottle of methylene blue,
light microscope
Method
Swab inside of mouth  Smear cotton bud on small area of slide  Add 3
drops of methylene blue to stain cell sample  Cover with cover slip 
View with microscope
Supervision level
The activity can be carried out by learners
without direct (one-to-one) supervision
YES
If NO, please list the elements that require direct supervision or handling by a
member of staff.
Risk assessment carried out by: …………………………… Date: ……………
Approved by teacher: ………………………...................... Date: ……………
30
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY B
Activity B: Liquids and solutions
Liquids and solutions A
A colorimetric method for estimating the concentration of starch in
solution
Iodine solution turns blue-black in the presence of starch.
Your task is to identify the concentration of starch in an unknown solution by
constructing a standard curve.
Method standard curve for iodine and starch
Equipment
0.5% starch solution
5 ml syringe
Distilled water
5 × 50 ml beakers
Marker pen
Using the stock 0.5% starch solution make serial dilutions of this to create 10
cm 3 of 0.4%, 0.3%, 0.2% and 0.1% solutions.
V1C1 = V 2C2
V 1 = volume of starting solution needed to make the new solution
C 1 = concentration of starting solution
V 2 = final volume of new solution
C 2 = final concentration of new solution
Example
Make 5 cm 3 of a 0.25M solution from 2.5 cm 3 of a 1 mol l –1 solution.
V1C1 = V 2C2
(V 1 ) (1 mol l –1 ) = (5 cm 3 × 0.25 mol l –1 )
V 1 = (5 cm 3 × 0.25 mol l –1 )/1 mol l –1
V 1 = 1.25 cm 3
So you will need to use 1.25 cm 3 of the 1 mol l –1 solution.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
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ACTIVITY B
Since you want the diluted solution to have a final volume of 5 cm 3 , you will
need to add (V 2 – V 1 = 5 cm 3 – 1.25 cm 3 ) = 3.75cm 3 of diluent.
Final starch concentration (%)
0.4 0.3 0.2 0.1
Volume of 0.5% starch solution
(cm3 )
Volume of distilled water (cm 3 )
Experimental phase
Equipment
Colorimeter
Cuvette holder
7 cuvettes
5 × 1 ml syringes
Cuvette cover film
Iodine solution
Starch solutions prepared as calculated above
1.
Construct a suitable table to record your data.
2.
Set the colorimeter to read transmission (T) and use the red LED (or
equivalent filter).
3.
Zero the colorimeter using a cuvette containing 1 cm 3 of distilled water
measured with a syringe and 2 drops of iodine. Cover the cuvette top
with cuvette film and invert the cuvette to ensure that the iodine has
mixed with the solution. Make sure that you do not touch the side of the
cuvette as this will leave oils and dirt from your fingers on the cuvette
and affect the percentage transmission.
4.
The colorimeter should read 100% transmission.
5.
Using a syringe take 1 cm 3 of 0.5% starch solution and add it to a
cuvette, then add 2 drops of iodine solution. Cover the cuvette top with
cuvette film and invert the cuvette to ensure that the iodine has mixed
with the solution. Make sure that you do not touch the side of the
cuvette as this will leave oils and dirt from your fingers on the cuvette
and affect the percentage transmission.
6.
Place the cuvette in the colorimeter and note down the percentage
transmission.
32
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY B
7.
Repeat this process using the starch solutions you have prepared. Be
careful not to cross-contaminate your sample and ensure you repeat the
measurement for each concentration.
8.
Calculate your averages.
9.
Construct a standard curve on graph paper.
10.
Record the percentage transmission of the unknown, making sure you
repeat your readings.
11.
Calculate your average percentage transmission for the unknown and
plot it on the standard curve to determine its concentration.
12.
Compare your results to those of the rest of the class and to the actual
concentration of unknown from your teacher.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
33
ACTIVITY B
Liquids and solutions B
Measuring the rate of amylase activity using a colorimeter
Starch is broken down to maltose by the enzyme amylase. The rate of this
reaction can be determined by measuring the change in percentage
transmission of a starch-amylase solution when exposed to iodine.
Iodine solution turns blue-black in the presence of starch.
As the amylase reacts with the starch the concentration of starch decreases
and the intensity of the colour change in the presence of iodine decreases.
This can be measured as an increase in percentage transmission over time
until there is no further increase in percentage transmission.
Apparatus
Colorimeter
Cuvettes
Cuvette holder
Iodine
Distilled water
Thermometer
Water bath set at 20°C
1 ml pipette
2 × 5 ml pipette
2 × 10 ml pipette
Pi pump
100 ml beaker
250 ml beaker
Boiling tubes
Boiling tube rack
Stopclock
100 ml measuring cylinder
0.5% starch
0.25% diastase (amylase)
Set up a colorimeter to percentage transmission (T) and the red LED (or
equivalent filter).
Prepare some diluted iodine solution by adding 60 cm 3 of distilled water to 3
cm 3 of bench iodine/potassium iodide solution and mixing well. This is the
solution that will be referred to simply as the ‘iodine solution’ in later
instructions.
34
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY B
Place 15 cm 3 of 0.5% starch solution in a boiling tube and put the tube in a
water bath at 20°C. Leave it to acclimatise.
Place 15 cm 3 of 0.25% diastase (amylase) solution in another boiling tube and
put this tube in the water bath to acclimatize.
Take a clean cuvette and, using a suitable graduated pipette, run in enough of
the iodine solution to approximately 3/4 fill the cuvette. Try to run in a whole
number of cm 3 of iodine solution as you will have to add the same volume of
iodine solution to your cuvettes in each subsequent stage of the experiment.
You will be adding 1 cm 3 of solution to this later so make sure there is
enough room for this in the cuvette as well. Make sure that you do not touch
the side of the cuvette as this will leave oils and dirt from your fingers on the
cuvette and affect the percentage transmission.
Use the cuvette containing the iodine as a ‘blank’ to calibrate your
colorimeter. Adjust the instrument so that the iodine solution produces a
reading of 100% transmission.
Obtain some more cuvettes and a stopclock. Discuss with your teacher how
many more cuvettes you will require to monitor the reaction until there is no
further change in percentage transmission.
Construct a suitable table to record your results.
Add the standard volume of iodine solution to each of the cuvettes in
readiness for the next stage of the experiment.
Now mix the acclimatised amylase solution with the acclimatised starch
solution. Immediately start the stopclock and then return the tube containing
the mixture to the water bath to keep it at a standard temperature.
After 30 seconds remove a 1 cm 3 sample of the mixture (using a graduated
pipette) and run it into the first prepared cuvette containing the iodine
solution. Quickly shake the cuvette to mix its contents and then place it in the
colorimeter and read off its percentage transmission. Record this data in your
table.
Take further 1 cm 3 samples of the amylase-starch mixture after 2 minutes and
then after suitable time intervals (which will depend on how much change
there has been between your 30-second reading and your 2–minute reading)
until no further changes in the percentage transmission readings occur. Ask
for advice at this stage if necessary.
Graph your results.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
35
ACTIVITY C
Activity C: Separation techniques
Cell fractionation and differential centrifugation of liver tissue
In order to study the function of a particular organelle it is often helpful to
isolate it from the rest of the cell. This can be done by cell fractionation.
1.
2.
3.
4.
5.
Tissue (eg liver) is placed in an ice-cold isotonic buffer. (The following
steps break the cells up rupturing membranes and bringing together
many chemicals that do not normally mix. The buffer minimises
unusual reactions, including self-digestion by lytic enzymes.)
Tissue cut into small pieces.
Tissue homogenised to break up whole cell.
Mixture filtered to remove debris.
Filtrate placed in centrifuge tubes and spun in a centrifuge.
The organelles will separate out according to their density and size. After a
time, the sediment (pellet) at the bottom of the centrifuge tube can be
separated from the supernatant (the liquid containing the remaining
organelles). The supernatant can then be spun again to separate the remaining
organelles. The exact times and speed for centrifugation vary from tissue to
tissue.
A bench-top centrifuge containing Eppendorf tubes.
36
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY C
Relative time to
separate
Organelle
Centrifuge
setting (g)
Time (min)
First to separate
Nuclei
800–1000
5–10
Mitochondria
10,000–20,000
15–20
Rough endoplasmic
reticulum
50,000–80,000
30–50
Plasma membrane
80,000–100,000
60
150,000–300,000
>60
Lysosomes
Smooth endoplasmic
reticulum
Last to separate
Free ribosomes
The diagrams shown below summarise the process (centrifugal force
measures the number of times that the force is greater than gravity.)
Five subcellular fractions (including the final resultant supernatant) can be
obtained and are shown as A, C, D, E and F in the diagr am. These fractions
can be investigated biochemically. For example, oxygen consumption or
hydrolytic enzyme activity can be measured.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
37
ACTIVITY C
Differential centrifugation (centrifugal fractionation) of liver tissue
Homogenised tissue
in sucrose solution
and buffer in
centrifuge tube
Centrifuge 10
min at 600 x g
Centrifuge 10 min
at 8500 x g
Centrifuge 100
min at 100 000 x
g
Fraction A
removed
Fraction B
removed
Fraction B re-suspended in
sucrose solution Centrifuge 100
min at 50000 x g
(a)
Fraction D
Fraction F
Fraction C
Fraction E
What is meant by homogenized?
___________________________________________________________
___________________________________________________________
(1)
38
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY C
(b)
The concentration of the suspending sucrose medium is chosen with
care. Briefly suggest why this is so.
___________________________________________________________
___________________________________________________________
___________________________________________________________
(1)
(c)
Suggest a reason for carrying out these procedures at ice -cold
temperature.
___________________________________________________________
___________________________________________________________
(1)
(d)
Predict which one of the fractions A, C, D, E or F is most likely to
contain mainly:
(i)
nuclei
___________________________________________________________
(1)
(ii)
ribosomes
___________________________________________________________
(1)
(e)
If lysosomes are marginally heavier than mitochondria, predict in which
fraction the lysosomes would most probably be found.
___________________________________________________________
(1)
(f)
(i)
Which fraction should show the highest rate of oxygen
consumption? Give a reason for your answer.
___________________________________________________________
___________________________________________________________
___________________________________________________________
(2)
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
39
ACTIVITY C
(ii)
Which fraction would you expect to produce most radioactively
labelled protein if labelled amino acids were added? Give a reason
for your answer.
___________________________________________________________
___________________________________________________________
___________________________________________________________
(1)
(iii) Which fraction should show the greatest amount of hydrolytic
enzyme activity? Give a reason for your answer.
___________________________________________________________
___________________________________________________________
___________________________________________________________
(2)
(iv) Which fraction should show the most evidence of synthesis of
messenger RNA? Give a reason for your answer.
___________________________________________________________
___________________________________________________________
___________________________________________________________
(2)
40
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY C
Differential centrifugation also plays an important role in our understanding
of DNA replication. The scientists Meselson and Stahl developed a simple
experiment to illustrate the mode of replication. Read on to find out how they
discovered the process.
The Meselson–Stahl experiment
This experiment used centrifugation in a density gradient to determine the
process by which DNA is replicated in the cell.
A molecule of DNA contains five different elements: C, O, H, P and N.
During DNA replication, a dividing cell uses sources of these elements from
its surroundings to assemble new copies of its DNA for use in the daughter
cells.
Nitrogen (N) exists naturally in two different isotopes: 14 N is the normal
isotope and 15 N is a heavy isotope that has an extra neutron. Cells grown in a
nutrient medium containing 15 N will use it to synthesise DNA that is heavier
than normal DNA containing only 14 N. Matt Meselson and Franklin Stahl
made use of this fact to investigate the way in which new copies of a cell’s
DNA are produced from existing ones.
In this worksheet you will recreate the Meselson –Stahl experiment using
different colours to simulate DNA strands synthesised from heavy and light
nitrogen. Choose one colour to represent a heavy DNA strand containing 15 N
and one to represent a light strand containing only 14 N. Colour in the boxes
below.
14
N DNA strand
15
N DNA strand
For the purposes of this worksheet, a molecule of DNA will be drawn as two
parallel lines.
Each line represents one of the strands of the double -stranded DNA molecule.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
41
ACTIVITY C
Using the colour scheme above, draw
1.
a DNA molecule which contains two strands of ‘heavy’ nitrogen DNA:
2.
a DNA molecule which contains two strands of ‘light’ nitrogen DNA:
3.
a DNA molecule which contains one strand of ‘heavy’ and one strand of
‘light’ nitrogen DNA:
Meselson and Stahl hypothesised that DNA replication could occur in one of
three ways:
1.
Conservative: The two strands of the parental DNA molecule remain
together and act as a template for a completely new double -stranded
molecule. The parental DNA molecule is conserved.
2.
Semi-conservative: The two strands of the ‘old’ parental DNA molecule
separate and each acts as a template for a new strand. At the end of
replication and cell division each new cell inherits a DNA molecule
consisting of one new and one old template strand.
3.
Dispersive: The parental DNA molecule is broken up into short
segments used as templates for the formation of new segments, which
are somehow joined together. At cell division, each new cell inherits a
DNA molecule with some old and some new nucleotides in each strand.
To determine which of these hypotheses was correct, Meselson and Stahl
grew bacteria in a heavy nitrogen medium for several generations, so that
virtually all their DNA contained 15 N. Next, they transferred the bacteria to a
nutrient medium containing 14 N. All ‘old’ strands of DNA existing before the
bacteria were transferred would be heavy 15 N stands whereas any ‘new’
strand of DNA synthesised after the transfer would be light. The scientists
removed samples of cells after there had been enough time to reproduce one,
two and three new generations. They then broke open the cells of each
generation, purified their DNA and used centrifugation to determine what
weights of DNA each cell contained. In centrifugation, heavier molecules
form bands near the bottom of the tube, whereas lighter molecules form bands
near the top.
42
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY C
1.
What the scientists actually saw in each centrifuge tube is displayed
below. What do these results imply about the mechanism of DNA
replication?
14N
14N
14N
15N/ 14N
15N/ 14N
15N/ 14N
15N
15N
15N
Parental DNA
2.
After 1
generation
After 2
generations
To confirm their results, the scientists allowed the cells to grow for
three generations after being removed from the 15 N to 14 N. In the space
below, draw (a) the eight DNA molecules and (b) the corresponding
centrifuge tube bands that would be expected after three rounds of the
14
N medium.
14N
15N/ 14N
15N
See the animation below to find out more:
http://www.sumanasinc.com/webcontent/animations/content/meselson.html
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
43
ACTIVITY C
Answers
Differential centrifugation
(a)
Break open
(b)
Osmotic effects = isotonic = reduced lysis of cell organelles
(c)
Reactions are slower
(d)
(i)
(ii)
(e)
C = lysosomes
(f)
(i)
(ii)
(iii)
(iv)
nuclei = A
ribosomes = F
D = mitochondria present respiration
F = ribosomes + protein synthesis + translation + tRNA
C = lysosomes present
A = nucleus + transcription
The Meselson–Stahl experiment
1.
Semi-conservative replication.
2.
The band at 14 N would become thicker and the band at
remain the same with each successive generation.
44
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
15
N/ 14 N would
ACTIVITY D
Activity D: Antibody techniques
Introduction
Enzyme linked immunosorbent assay (ELISA) is a system that is used to
detect specific antigens or antibodies. It can exist in two forms, one which
detects the presence of a particular antibody and another which detects the
presence of a specific antigen. The following diagram shows a specific
antibody to a disease antigen bound to an assay well being used to detect
specific antigens in solution. A second antibody is added which is conjugated
to an enzyme. The enzyme will be active on a dye -based substrate. When the
bonds in the substrate are broken down dye is liberated, indicating the sample
wells that contain antigen specific to the detection antibody. This is known as
an antigen-capture ELISA.
Another version detects antibody by binding antigen to the plates and then
applying, for example, blood serum. If the serum contains specific antibody
this will bind to the antigen. This is called an antibody -capture ELISA. A
second detector antibody bound to an enzyme is then applied which
recognises antibody. Substrate is added as above and a positive result
detected by dye production.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
45
ACTIVITY D
The ELISA test was one of the first sensitive tests used to detect HIV
infection by recognising circulating antibody in a potentially infected
patient’s serum. This does not indicate virus but antibodies to the virus. To
detect current virus production an ELISA was developed where antibody to
p24 capsid antigen was bound to an ELISA plate. The detection of circulating
viral antigen indicates active infection and virus production.
Demonstration of ELISA in the laboratory
Commercial ELISA kits are available to detect a wide variety of subst ances,
conditions and diseases.
Bio-Rad Biotechnology Explorer™ ELISA Immuno Explorer Kit
For classroom use the Bio-Rad Biotechnology Explorer™ ELISA Immuno
Explorer Kit is a useful tool to demonstrate the technique. (Bio-Rad
Biotechnology Explorer™ ELISA Immuno Explorer Kit. Catalogue No. 166 2400EDU)
Relevant literature and prices are available online at www.explorer.biorad.com. It is recommended that teachers access the Biorad education site and
select Classroom Kits from the pull-down Catalog Index box. Please note
literature is free to download but educators have to register on the site. An
overview of the kit appears on the catalogue page.
Each kit is designed to allow the use of three distinct protocols with the same
reagents:
1.
2.
3.
ELISA for tracking disease outbreaks
antigen detection ELISA
antibody detection ELISA.
The kit contains uncoated ELISA strips that can b e coated with the primary
antibody (rabbit-antichicken) to provide an antigen-capture assay, eg Protocol
2, or coated with antigen (chicken immunoglobulin) to act as an antibody
capture, eg Protocol 3, assay. In both cases a goat anti -rabbit antibody
conjugated to horseradish peroxidase is used as the secondary detector
antibody.
The kit has enough reagents to run 12 workstations with up to four learners
per workstation. Everything required for the experiments is contained within
the kit with the exception of 50 μl pipettes, variable microlitre pipettes and
basic laboratory glassware and reagents.
46
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY D
NCBE provide Volac Minipipetts fixed 50 μl for £16.00/each.
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/PDF/NCBEpricelist.pdf .
SAPS ELISA kit for Botrytis
A low cost ELISA kit developed by SAPS can be used to detect the fungal
pathogen Botrytis. This fungus commonly infects plant material such as
strawberries, raspberries, tomatoes and flowers. The kit contains all the
specialised equipment together with the necessary antibodies, the substrate
and a Botrytis culture for reference. The materials provided are sufficient for
5 groups of students.
Further information is available at the following webpage
http://www.saps.org.uk/secondary/teaching-resources/120-the-saps-elisa-kitfor-botrytis.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
47
ACTIVITY E
Activity E: Microscopy
Aim
To estimate the total and viable (living) number of yeast cells in culture using
a haemocytometer.
Background
A haemocytometer was traditionally used to estimate blood counts. It consists
of a glass slide with a finely etched series of grids on the su rface.
The centre grid is 1 mm × 1 mm subdivided into 25 smaller squares in the
centre.
The double Neubauer slide shows a square of nine large 1 mm × 1 mm etched
grids per chamber, as shown below. A double Neuba uer slide contains two
chambers to allow (pseudo)replicate counts to be made from a single slide .
When a specially strengthened cover slip is placed over the grid a gap of 0.1
mm is left between the grid and the sample.
The volume over one large grid is 0.01 cm × 0.1 cm × 0.1 cm = 0.0001 cm 3 .
This figure can be used to estimate the number of cells/cm 3 .
Large cells such as yeasts and other eukaryotic cells can be easily counted
using the 1 mm × 1 mm grids. If counting smaller cells such as algae and
bacteria, the smaller ruled centre grid shou ld be used with the appropriate
scale factor when estimating cells/cm 3 .
48
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY E
Viable counting of yeast
It is often important in growth studies to estimate the number of living cells
within a cell culture. This requires the use of a vital stain, i .e. one that only
stains either living or dead cells. Methylene blue dye is often used because it
only stains dead cells. In yeast and mammalian cell cultures it stains dead
cells blue; living cells are unstained. This gives a simple method for
establishing a viable cell count as compared to a total cell count.
Materials
Yeast culture (0.5 g of dried yeast resuscitated in 100 ml 1% glucose solution
for 15 minutes)
Double/single Neubauer haemocytometer or equivalent
Two tally counters per learner
Microscope (with x40 objective, x10 eyepiece)
0.1% methylene blue
Method
Add equal volumes of 1% methylene blue and yeast culture. Leave for 5
minutes but complere the cell count within 10–15 minutes.
Clean the haemocytometer slide with lens tissue and alcohol. Fix the cover
slip over the etched grids by moistening the slide with breath or a tiny drop of
distilled water.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
49
ACTIVITY E
Make sure the cover slip is firmly stuck to the glass. Interference fringes
should be visible when looking through the sides of the cover slip.
Note: Only use haemocytometer cover slips, as standard cover slips will not
work as they deform and may also break.
Gently fill each chamber with the methylene blue/yeast culture mixture using
a dropper or Pasteur pipette. View at x400 magnification. If the culture is too
numerous to count use serial dilution before counting. Remember to take
account of any dilution factor when estimating the cell count in your initial
culture.
Repeat and average readings before estimating cell density per millilitre as
follows:
 Assuming a non-clumped suspension of cells count the cell number in each
of the four corner squares and the centre, i .e. 5 × 1 mm 2 squares
 This gives you a count of cells per 0.5 mm 3 . Multiply this by 2 to express
in cells/mm 3 . Then multiply by 1000 to determine cell count per cm 3 (ml).
Extension
The above basic technique can be used in a number of potential Advance d
Higher Biology projects involving yeast. For example, the potential
antifungal effect of metals ions such as Zn 2+ and Cu 2+ could be investigated
through the addition of zinc chloride or copper sulphate solutions to yeast
cultures is one possibility.
SAPS (Science and Plants for Schools) have also proposed potential projects
on the effects of increased salt concentrations on autolysis in yeast cultures.
References
http://www.saps.org.uk/secondary/teaching-resources/112-testing-viabilityof-yeast-at-different-stages-of-the-autolysis-process
or
http://www.nationalstemcentre.org.uk/elibrary/file/3117/yeast.pdf
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UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY F
Activity F: Aseptic technique
Practising aseptic transfer technique: a simulation of liquid-toliquid subculturing
Subculturing involves the removal of microorganisms (the inoculum) from a
source and transferring them to a fresh medium (the inoculate). It is very
important that the transfer is carried out using good aseptic technique so that
the medium is not contaminated with unknown microorganisms from the
environment.
In this activity you will practice a liquid-to-liquid transfer using a pipette.
The aim is find out how efficient your aseptic transfe r technique is. You will
transfer sterile nutrient broth rather than known microorganisms from a
culture. Any growth that occurs in your broths will be due to contamination
from microorganisms in the environment.
Lesson 1
Materials required per learner
6 universal bottles of sterile nutrient broth (9 ml per bottle)
6 sticky paper labels and permanent market pen
Air displacement pipette (set to 1 ml) and 4 sterile pipette tips OR 4 sterile
1 ml plastic dropping pipettes
Bunsen burner and heatproof mat
100 ml 1% bleach solution and paper towel
Discard jar containing 100 ml 1% Virkon solution
Method
Preparation
1.
Clear the place where you are going to work of all items.
2.
Prepare yourself: tie back long hair, wash and dry hands, cover cuts
with plasters, wear lab coat, safety goggles and gloves.
3.
Collect all the materials you will need at your workspace.
4.
Prepare your workspace: disinfect the surface with half of your bleach
solution and paper towel, place the Bunsen on the heat mat in a central
location and set it to the blue flame.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
51
ACTIVITY F
5.
6.
Put the bottles next to the heat mat on the left and the discard jar,
pipette and pipette tips on the right (use the opposite arrangement if
you’re left handed).
Label five of the bottles 1–5, label the other bottle control. You should
also record the type of broth (NB), your initials and the date. The
control bottle will not be opened.
Aseptic transfer (instructions for right-handed people)
1.
Carry out all work within a 20-cm radius in front of the bunsen.
2.
Loosen the lids on bottles 1–5 to allow easy removal.
3.
Take the pipette and tip/plastic dropping pipette in your right hand.
4.
Hold bottle number 1 in your left hand.
5.
Remove the lid with the little finger of your right hand and hold on to
it. Turn the bottle rather than the lid.
6.
Immediately flame the bottleneck by passing it through the flame.
7.
Squeeze the pipette before it enters the broth and remove 1 ml of broth.
8.
Flame the bottleneck again.
9.
Replace the lid and put the bottle back onto the work surface.
10. Repeat steps 4–6 to open and flame bottle 2.
11. Transfer the broth in the pipette into bottle 2.
12. Repeat steps 8 and 9.
13. Discard the pipette tip/dropping pipette in the Virkon jar.
14. Repeat steps 3–13 to transfer 1 ml of broth from tube 2 to 3, then 3 to 4
and finally 4 to 5. Use a new pipette tip/sterile dropping pipette for
each transfer.
15. Incubate all bottles at 30°C for 48 hours.
Clearing up
1.
Turn off the bunsen.
2.
Clear away the bunsen, heat mat and air displacement pipette.
3.
It is good practice to leave the pipette tips/plastic dropping pipett es in
the Virkon for 24 hours before disposing of them.
4.
Disinfect the work surface with the remainder of the bleach and paper
towels.
5.
Remove your safety goggles, lab coat and gloves.
6.
Wash and dry your hands thoroughly.
Thinking point: How do Aseptic transfer steps 1, 6, 7 and 8 help to increase
the efficacy of the aseptic transfer?
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UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
ACTIVITY F
Lesson 2
1.
2.
3.
4.
Remove broths from incubator. Do not open them at any point.
Compare the appearance of the broths in bottles 1 –5 with the broth in
the control bottle.
Check your broths for contamination and record your observations in
the table on the next page. Your teacher should also check your broths
and sign off the table if they agree with your observations.
The bottles of broth should be returned to your teacher/lab technician to
be autoclaved.
UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012
53
ACTIVITY F
Examination of sterile media broths
Name: _____________________________________________________
Date prepared and
incubated
Broth number
Date checked
Does broth
appear clear (c)
or turbid (t)?
Temperature
of incubator
at start (°C)
Are
microorganisms
growing in
broth?
Temperature
of incubator
at end (°C)
Is broth sterile
or
contaminated?
1
2
3
4
5
Control
Number of sterile broths:
Signature of checker:
The further along the series that your broths are contamination-free, the better
your aseptic technique.
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UNIT 1 (AH, BIOLOGY)
© Crown copyright 2012