eibe unit 1 : english language version - ipn

Contributors to this UnitEckhard R. Lucius (Unit Co-ordinator)

Catherine Adley, Jan Frings, Cecily Leonard, Dean Madden, Uta Nellen,

Patricia Nevers, John W. Schollar, Marleen van Strydonck, Paul E.O. Wymer.

UN

IT1Microbesand molecules

European Initiative for Biotechnology Education

EIBE

This teaching package (EIBE Unit 1) was produced by the European Initiative for Biotechnology Education. The Unit is available on the World Wide Web, at: http://www.reading.ac.uk/NCBE For more information about this Unit , please contact: Eckhard R. Lucius, IPN, Universität Kiel, Olshausenstraße 62, D-24098 KIEL 1. Tel: + 49 (0) 431 880 3137 Fax: + 49 (0) 431 880 3132 E-Mail: [email protected] Copyright (c) 1995, 1997

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2 UNIT 1: MICROBES AND MOLECULES EIBE European Initiative for Biotechnology Education 1995

The European Initiative for Biotechnology Education (EIBE)seeks to promote skills, enhance understanding and facilitateinformed public debate through improved biotechnologyeducation in schools and colleges throughout the EuropeanUnion (EU).

EIBE Co-ordinatorHorst Bayrhuber, Institut für die Pädagogik der Naturwissenschaften an der Universität Kiel, Olshausenstraße 62,D-24098 KIEL 1, Germany. Telephone: + 49 (0) 431 880 3137 (EIBE Secretary: Regina Rojek). Facsimile: + 49 (0) 431 880 3132.

EIBE ContactsAUSTRIA❙ Rainhart Berner, Höhere Bundeslehr- und Versuchsanstalt für Chemische Industrie Wien, Abt. für Biochemie,Biotechnologie und Gentechnik, Rosensteingasse 79, A-1170 WIEN.

BELGIUM❙ Vic Damen / Marleen van Strydonck, R&D Groep VEO, Afdeling Didaktiek en Kritiek, Universiteit vanAntwerpen, Universiteitsplein 1, B-2610 WILRIJK.

DENMARK❙ Dorte Hammelev, Biotechnology Education Group, Foreningen af Danske Biologer, Sønderengen 20, DK-2860 SØBORG.❙ Lisbet Marcussen, Biotechnology Education Group, Foreningen af Danske Biologer, Lindevej 21, DK-5800 NYBORG.

EIRE❙ Catherine Adley / Cecily Leonard, University of Limerick, Plassey, LIMERICK.

FRANCE❙ Gérard Coutouly, LEGPT Jean Rostand, 18 Boulevard de la Victorie, F-67084 STRASBOURG Cedex.❙ Laurence Simonneaux, Ecole Nationale de Formation Agronomique, Toulouse-Auzeville, Boîte Postale 87,F-31326 CASTANET TOLOSAN Cedex.

GERMANY❙ Horst Bayrhuber / Eckhard R. Lucius / Regina Rojek / Ute Harms / Angela Kroß, Institut für die Pädagogik derNaturwissenschaften, Universität Kiel, Olshausenstraße 62, D-24098 KIEL 1.❙ Ognian Serafimov, UNESCO-INCS, c/o Jörg-Zürn-Gewerbeschule, Rauensteinstraße 17, D-88662 ÜBERLINGEN.❙ Eberhard Todt, Fachbereich Psychologie, Universität Gießen, Otto-Behaghel-Straße 10, D-35394 GIEßEN.

ITALY❙ Antonio Bargellesi-Severi / Stefania Uccelli / Alessandra Corda Mannino, Centro di Biotechnologie Avanzate,Largo Rosanna Benzi 10 , I-16132 GENOVA.

LUXEMBOURG❙ John Watson, Ecole Européenne de Luxembourg, Département de Biologie, 23 Boulevard Konrad Adenauer,L-1115 LUXEMBOURG.

THE NETHERLANDS❙ David Bennett, Cambridge Biomedical Consultants, Schuystraat 12, NL-2517 XE DEN HAAG.❙ Fred Brinkman, Hogeschool Holland, Afdeling VP&I, Postbus 261, NL-1110 AG DIEMEN.❙ Guido Matthée, Hogeschool van Arnhem en Nijmegen, Technische Faculteit, HLO, Heijendaalseweg 45,NL-6524 SE NIJMEGEN.❙ Liesbeth van de Grint / Jan Frings, Hogeschool van Utrecht, Educatie Centrum voor Biotechnologie, FEO,Afdeling Exacte Vakken, Biologie, Postbus 14007, NL-3508 SB UTRECHT.

SPAIN❙ Maria Saez Brezmes / Angela Gomez Niño, Facultad de Educación, Universidad de Valladolid,Geologo Hernández Pacheco 1, ES-47014 VALLADOLID.

SWEDEN❙ Margareta Johanssen, Föreningen Gensyn, PO Box 37, S-26800 SVALÖV.❙ Elisabeth Strömberg, Östrabo Gymnasiet, PO Box 276, Kaempegatan 36, S-45181 UDDEVALLA.

THE UNITED KINGDOM❙ Wilbert Garvin, Northern Ireland Centre for School Biosciences, NIESU, School of Education, The Queen’s University ofBelfast, BELFAST, BT7 1NN.❙ John Grainger / John Schollar / Caroline Shearer, National Centre for Biotechnology Education, The University of Reading,PO Box 228, Whiteknights, READING, RG6 6AJ.❙ Jill Turner, Department of Science and Technology Studies, University College London, Gower Street, LONDON, WC1 6BT.❙ Paul Wymer, The Wellcome Centre for Medical Science, The Wellcome Trust, 210 Euston Road, LONDON, NW1 2BE.

3EIBE European Initiative for Biotechnology Education 1995 UNIT 1: MICROBES AND MOLECULES

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UN

IT1Microbes andmolecules

World Wide Web★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

❙ Safety, copyright andacknowledgements 4

❙ About this UnitIntroduction 5

❙ Model making Cutout DNA model 6 Cutout bacteriophage model 8

❙ DNA extraction Extracting DNA from bacteria 10 Extracting DNA from onions 12

❙ Productive microbes Amylase production 14 Cellulase production 16 Antibiotic production 18

❙ Monitoring microbial activity Bread dough 20

Immobilised yeast cells 22The microbial fuel cell 25

❙ Gene transfer Bacterial conjugation 27 Gene transfer by

Agrobacterium tumefaciens 32

❙ Appendix 1Recipes for microbiological media 35

❙ Appendix 2Basic microbiological techniques 36

Contents★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

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European Initiative for Biotechnology Education

Few areas are developing as rapidly asbiotechnology. So that they can be revised andkept up-to-date then distributed at minimum cost,the EIBE Units are published electronically.

These pages (and the other EIBE Units) areavailable throughout Europe and the rest ofthe world on the World Wide Web. They canbe found at:

http://www.reading.ac.uk/NCBE

All of the EIBE Units on the World WideWeb are Portable Document Format (PDF)files. This means that the high-qualityillustrations, colour, typefaces and layout ofthese documents will be maintained,whatever computer you have (Macintosh -including Power PC, Windows, DOS or Unixplatforms).

PDF files are also smaller than the files fromwhich they were created, so that it will take lesstime to download documents. However, to viewthe EIBE Units you will need a suitable copy ofthe Adobe Acrobat ® Reader programme.

The Acrobat ® Reader programme is availablefree-of-charge in several languages (Dutch,UK English, French, German and Italian). Itcan be downloaded from the EIBE or Adobeweb sites:

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With this software, you can view or print theEIBE Units. In addition, you will be able to‘navigate’ around and search the documentswith ease:.

PLEASE NOTE: Adobe and Acrobat are trademarks ofAdobe Systems Incorporated, which may be registeredin certain jurisdictions. Macintosh is a registeredtrademark of Apple Computer Incorporated.

http://www.reading.ac.uk/NCBE

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SafetyIn all of the EIBE Units, we have tried to check thatall recognized hazards have been identified and thatsuitable precautions are suggested.

Where possible, the proposed procedures are inaccordance with commonly-adopted general riskassessments. If a special risk assessment may benecessary, this has been indicated. Appendix 2 in thisUnit gives additional safety guidelines.

However, users should be aware that errors andomissions can be made, and that different employersand educational authorities adopt different standards.Therefore, before doing any activity, users shouldalways carry out their own risk assessment. Inparticular, any local rules issued by employers oreducational authorities MUST be obeyed, whatever issuggested in the EIBE Unit.

Unless the context dictates otherwise, it is assumedthat:

● practical work is carried out in a properlyequipped and maintained science laboratory;

● any mains-operated equipment is properlymaintained;

● care is taken with normal laboratory operationssuch as heating substances;

● good laboratory practice is observed whenchemicals or living organisms are used;

● eye protection is worn whenever there is anyrecognised risk to the eyes;

● pupils and/or students are taught safe techniquesfor activities such as handling chemicals andmicroorganisms.

Development team● Catherine Adley

The University of Limerick, Eire.● Jan Frings

Hogeschool van Gelderland, The Netherlands.● Cecily Leonard

The University of Limerick, Eire.● Eckhard R.Lucius (Unit Co-ordinator)

Germany.● Marleen van Strydonck

The University of Antwerp, Belgium.● Paul E.O. Wymer

The Wellcome Trust for Medical Science,London, The United Kingdom.

Design, illustration, typesetting, additional text

and editing: Dean Madden, NCBE, The Universityof Reading, The United Kingdom. Illustrations andtypography Copyright © Dean Madden, 1997

AcknowledgementsEIBE is especially grateful to the following people for their help in the production of these materials. Liesbeth van deGrint (Hogeschool van Utrecht, The Netherlands) and John Schollar (National Centre for Biotechnology Education, TheUniversity of Reading, The United Kingdom) arranged and ran a multinational workshop on which the materials in this Unitwere tested. The following teachers took part and gave many helpful comments on the draft materials: E. Vergauts, L. Daniels,L. Neels (Belgium); Dorte Hammelev (Denmark); Lucienne Diemer, Gérard Coutouly (France); Thomas Jeß, Dr U. Schnack;Dr E. Lipkow (Germany); A. de Graaf, Guus Smid, J. Gradener (The Netherlands); Rebecca Weston, Jane Gent, Derek Mackie,Sarah Whitethread, Maggie Parson (The United Kingdom).

© CopyrightThis EIBE Units is copyright. The contributors to thisUnit have asserted their moral rights to be identified ascopyright holders under Section 77 of the Designs,Patents and Copyright Act, UK (1988).

Educational use. Electronic or paper copies of thisEIBE Unit, or individual pages from it may be madefor classroom use, provided that the copies aredistributed free-of-charge or at the cost of

reproduction, and that the contributors to the Unit arecredited and identified as the copyright holders.

Other uses. The Unit may be distributed byindividuals to individuals for non-commercial purposes,but not by means of electronic distribution lists,mailing (listserv) lists, newsgroups, bulletin board orunauthorised World Wide Web postings, or other bulkdistribution, access or reproduction mechanisms thatsubstitute for a susbscription or authorised individualaccess, or in any manner that is not an attempt in goodfaith to comply with these restrictions.

Commercial use. The use of materials from this Unit forcommercial gain, without the prior consent of thecopyright holders is strictly prohibited. Should you wishto use this material in whole or in part for commercialpurposes, or to republish it in any form, you should contact:

EIBE Secretariat (Regina Rojek)c/o Institut für die Pädagogik

der NaturwissenschaftenUniversität KielOlshausenstraße 62D-24098 KIEL 1Germany

Telephone:+ 49 (0) 431 880 3137Facsimile: + 49 (0) 431 880 3132E-Mail: [email protected]


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About this UnitThis Unit comprises a collection ofactivities designed to be used independentlyor in series as part of a teachingprogramme. They have been devised bypractising teachers and educationalists fromseveral European countries, broughttogether with support and encouragementfrom DGXII of the EuropeanCommission, under the auspices of EIBE,the European Initiative for BiotechnologyEducation.

All of the activities have been extensivelytested on practical workshops involvingteachers from across Europe.

The activities in this Unit consist of:

1. Inexpensive models of a DNAmolecule and various microorganisms.These are intended to show the essentialfeatures of these systems and to providean opportunity for students to considerof the relative sizes of microbes.

2. Simple, safe and inexpensive methodsof extracting DNA, to give studentsexperience of the basic techniquesinvolved and allow them to see thegenetic material.

3. A range of investigations emphasizinga) the presence of microorganisms inthe environment and the selection ofuseful strains and; b) some of theproducts that microbes can provide(enzymes and antibiotics). Two of theseinvestigations are qualitative; oneprovides a quantitative assay of enzymeproduction.

4. Several investigations of the effects ofmicrobial growth, ranging from simplework with bread dough, through workon fermentation and finally the directmeasurement of metabolic activity ofyeast. In each of these investigationsthere is ample opportunity for further,extended investigations. To motivate

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Nstudents, activities have been selected toreflect biotechnological applicationsrather than theoretical principles alone.

5. Two demonstration activities whichshow natural methods of gene transfer,intended as a practical introduction tothe principles of genetic modification.

Because two of the practical activitiesinvolve the production and action ofantibiotics and the transfer of antibioticresistance, additional information regardingthese topics is included.

The authors have tried to provide acombination of both simple and moreadvanced activities, in the hope that anybiology teacher will find something ofinterest and value amongst them. Twoappendices provide some basic informationabout the use of microbiological techniquesin the school laboratory.

In the near future, supplementary guideswill be available, providing details ofcurriculum links, safety regulations, theavailability of materials and other relevantinformation in different parts of theEuropean Union.

Comments on these materials are verywelcome, especially from teachers, at whomthey are principally aimed. Comments andqueries about this Unit should be sent to:

Eckhard R. LuciusInstitut für die Pädagogik

der NaturwissenschaftenUniversität KielOlshausenstraße 62D-24098 KIEL 1Germany

Telephone: + 49 (0) 431 880 3137Facsimile: + 49 (0) 431 880 3132E-Mail: [email protected]

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6 UNIT 1: MICROBES & MOLECULES EIBE European Initiative for Biotechnology Education

Cut-out DNAmodel

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Aim● To show the essential features of the

structure of DNA (deoxyribonucleic acid).

OrganisationIt takes about 30 minutes to make thismodel. If the cut-outs (opposite) have alsoto be coloured-in, allow more time.

Equipment and materialsRequired by each student or group of students

● Scissors● Strong paper glue, adhesive tape or a

small stapler. Note: double-sided tape issatisfactory, although it tends to come unstuckafter some time.

● Thread (to hang label from model, andto hang model up when completed)

Note: to provide a sturdier model, two sets of the sugar-phosphate ‘backbones’ can be glued together, back-to-back.

Assembly details1. Cut out the sugar/phosphate ‘back-

bones’ A and B.2. Cut out the base pair ‘rungs’.3. Fold down the tabs on the rungs as

shown opposite.4. Stick the tab on one end of each rung

onto the numbered boxes along back-bone A. You can attach the rungs in anyorder (pointing either way).

5. Stick the tabs on the other end of therungs to the corresponding numbers onbackbone B.

6. Attach the label to the double helixmodel, using thread. The diagramsbelow could also be used to label themodel, if desired.

AcknowledgementThis model is adapted from one devised bythe government research organisationCSIRO, in Australia, for their ‘DoubleHelix’ Science Club. EIBE is grateful toCSIRO for the original idea and forpermission to adapt their model.

sugar

base

G

A

T

C

G

G

T

A

G

C

C

phosphate

C

A T

nucleotide

The structure of DNAChains of sugar and phosphate molecules form thetwo ‘backbones’ of DNA. Between these, four bases:thymine (T); cytosine (C); adenine (A) and guanine(G) are linked by hydrogen bonds.

PHE

LEU

SER TYR

STOP

CYS

TRP

PHE

LEU

SERSERSER

TYR

STOP

CYSSTOP

PRO HIS

GLN

ARGLEULEULEULEU

PROPROPRO

HIS

GLN

ARGARGARG

ILE

MET

THR ASN

LYSILEILE

THRTHRTHR

ASN

LYS

SERSERARGARG

VAL ALA ASP

GLU

GLYVALVALVAL

ALAALAALA

ASP

GLU

GLYGLYGLY

TCAG

TCAG

TCAG

TCAG

TCAG

T C A G

Firstposition

Secondposition

Thirdposition

The genetic codeEach sequence of three bases on the DNA doublehelix encodes one of twenty amino acidsrepresented in the body of the table by three-lettercodes.

European Initiative for

BiotechnologyEducation

DNAMODEL

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Model microbes★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

It is often difficult for students to

appreciate the scale of microorganisms.

In addition, the three-dimensional

structure of microbes is difficult for

students to understand from a two-

dimensional view down a microscope.

By making models, this activity helps

students to appreciate both the main

features of and the relative sizes of

microbes.

This activity is more successful if

students are given freedom to devise their

own models, rather than following a

specified plan. Consequently, this task

may be better completed at home (where

a wider range of materials and more time

may be available).

Note: for those misguided individuals who

believe that model-making is beneath their

dignity, it may be necessary to point out the

long and distinguished history of model-

making in molecular biology —although

nowadays, physical models have been largely

replaced by their computer counterparts.

Aim● To show the essential features of the

structure of a lambda bacteriophageand a variety of bacteria.

● To help students to appreciate therelative sizes of viruses and bacteria.

OrganisationIt takes about 30 minutes to make thebacteriophage model. If the net (opposite)has also to be coloured-in, assembly willtake longer. Depending upon the caretaken, the bacteria models may take an houror more to constract. Note: This activity mayusefully be set as homework.

Equipment and materialsRequired by each student or group ofstudents● Text books, showing the structure of

viruses and bacteria

● A selection of model-making materialse.g. card, discarded packaging, table-tennis balls, beads etc.

● Scissors● Glue, adhesive tape or a small stapler● Paints

Instructions to studentsVirus

Use the cutout net provided to make amodel of a lambda bacteriophage.

Bacteria

Use the materials provided to make modelsof bacteria (rods and cocci).

For each of your models:

1. Identify the basic features that areshown e.g. cell membranes, geneticmaterial;

2. Work out the size of the organism yourmodel represents;

3. Think of a simple way of explaining therelative sizes of the models to pupilsyounger than you e.g. ‘if real bacteriawere this size, you would (bycomparison) be as tall as a house’.

ExtensionStudents could also be asked to makemodels of plant and/or animal cells. Thesecould be used to reinforce the differencesbetween prokaryotes and eukaryotes, and toemphasize the serial endosymbiosishypothesis (explaining the supposedevolutionary origin of eukaryotes).

Additional informationModel-making activities are also describedin the following publication:

Nicholl, L. and Nicholl, D. (1987)Modelling the eukaryotic chromosome:a stepped approach. Journal of BiologicalEducation 21 (2) 99–104.


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Net for head of bacteriophage λ

Photocopy this net onto card or acetate sheet (O

HP

film).

Cut round the outside of the net, score and fold on all lines,

then glue the tabs with a strong, quick-drying paper adhesive.

Head,

containingD

NA

Tail

The DN

A strand can be represented by

thick thread or wire.

Use a drinking straw

to make the tail

(the flexible type with concertations,

stretched out, make the m

odel more

realistic).

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Isolation of DNAfrom bacteria

Procedure1. Prepare a culture of Escherichia coli K-12, at

least 4 days before you wish to isolate theDNA.

2. Add a small quantity of lysozyme to 5 mlof bacterial suspension and mix well.

3. Incubate the mixture for 30 minutes at 37 °C.4. Add 0.5 ml of household detergent to the

bacterial suspension.5. Incubate the mixture for 2 minutes at

60 °C in a beaker of water.6. Allow the mixture to cool in cold water

for a few minutes.7. Slowly and very carefully pour a layer of

cold ethanol onto the surface of themixture.

8. The nucleic acids (DNA and RNA) willprecipitate into the upper (ethanol) layer.

Extension1. The DNA may be stained e.g. using

aceto-orcein or methylene blue solution.

Safety precautionsStandard microbiological safety proceduresshould be followed when handling cultures. Careshould be taken when using hot water (step 5).

AcknowledgementThis procedure is based on one devised byHertel et al. and Süßmuth et al. (1987) whoisolated DNA from Bacillus subtilis. Wewould like to thank Professor JosephLengeler of Osnabrück for the suggestionof using household detergent for this work.

plasmids,small rings of DNA

chromosome

cell wall

cytoplasm

cell membrane

ribosomes,sites of protein

synthesis

A typical bacterial cell, showing thelocation of the nucleic acids, and wherethe lysozyme and detergent act (on thecell wall and membrane).

This activity uses lysozyme (an enzyme)

to degrade the cell walls of bacteria and

household detergent to disrupt the inner

cell membranes, releasing nucleic acids

(DNA and RNA).

Aim● To isolate nucleic acids from the

bacterium Escherichia coli K-12.

Advance preparationCultures of bacteria, grown on nutrientbroth, must be prepared at least 4 days inadvance (only mature cultures yieldsignificant quantities of nucleic acids).

OrganisationThis activity takes about 50 minutes tocomplete, including a period of 30 minutesduring which the bacteria are incubatedwith the enzyme.


● Mature culture of Escherichia coli K-12(see Advance preparation, above)

● Lysozyme powder - only the smallestamount that can be held on the tip of aspatula is required

● Cold ethanol, 6 ml (IMS is satisfactory)● Household detergent e.g. Pril (Henkel), Woolite

(Reckitt & Colman), 0.5 ml● Inoculation loop● Distilled water, 3 ml● Test tubes, 2● Pipette or 1 ml plastic syringe, for

dispensing water and lysozyme solution● Water bath, set at 60 °C● Incubator, set at 37 °C

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1. Prepare aculture of E.coliK-12 on nutrientbroth.

2. Add a smallquantity oflysozyme to 5 mlof the bacterialsuspension, andmix well.

3. Incubate themixture for 30minutes at 37 °C.

4. Add 0.5 ml ofhouseholddetergent to thebacterialsuspension.

5. Incubate themixture for 2minutes at 60 °Cin a beaker ofwater.

6. Allow themixture to coolin cold water fora few minutes.

7. Slowly and verycarefully pour alayer of coldethanol onto thesurface of themixture.

8. The nucleicacids (fine whitestrands) willprecipitate intothe upper(ethanol) layer.

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DN

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Isolation of DNAfrom onions

Procedure

1. Add the table salt to the washing-up liquid.Make up to 100 ml with distilled water.

2. Chop the onion into small pieces, but nosmaller than 10 mm x 10 mm.

3. Add the chopped onion to a beaker withthe salty washing-up liquid solution.

4. Put the beaker in a water bath at 60 °C forexactly 15 minutes.

5. Cool the mixture by standing the beaker inan ice water bath for 5 minutes, stirringfrequently.

6. Pour the mixture into a blender and blendit for no more than 5 seconds.

7. Filter the mixture into a second beaker.Ensure that the foam on top of the liquiddoes not contaminate the filtrate.

8. Add 2–3 drops of protease to about 6 mlof the onion tissue extract in a boilingtube and mix well.

9. Pour ice cold ethanol carefully down theside of the boiling tube, to form a layer ontop of the onion extract. Leave the tubefor a few minutes without disturbing it.

10. Nucleic acids will precipitate into theupper (ethanol) layer.

Extension1. The DNA may be stained e.g. using

aceto-orcein or methylene blue solution.2. DNA may also be extracted from certain

animal tissues (e.g. cod roe, liver, calfthymus i.e. sweetbread). Here, a mortarand pestle (with silver sand) instead of ablender provides a gentler method ofbreaking up the tissue, ensuring that theDNA is not sheared.

Safety precautionsThis procedure presents no particular safetyhazards, although care should be exercisedwhen chopping the onion or using the blender.

AcknowledgementThis method is adapted from A Sourcebook ofBiotechnology Activities by Alison Rasmussen andRobert Matheson (1990) National Associationof Biology Teachers / North CarolinaBiotechnology Center. ISBN: 0 941212 09 2.

The full publication is available from theNABT, 11250 Roger Bacon Drive #19,Reston, Virginia 22090, USA.

This is a crude method of isolating DNAand RNA from plant tissue. First, the tissueis broken up mechanically. Householddetergent is used to degrade both the cellmembranes and those surrounding thenuclei. Cell fragments are separated byfiltration; the nucleic acids and solubleproteins remain. An enzyme is used todegrade the proteins, then the nucleicacid is precipitated into ice cold ethanol.

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Aim● To isolate nucleic acids from onion tissue.

Note: nucleic acids prepared in this way will notbe very pure. The essential purpose of thetechnique described is to demonstrate the majorprinciples involved in the extraction of DNAfrom tissue.

Advance preparationThe ethanol used must be ice cold. Place it ina plastic bottle in a freezer at least 24 hoursbefore you attempt this activity.

OrganisationThis activity takes about 35 minutes, includingan incubation period of 15 minutes.


● Domestic food blender● Sharp vegetable knife and chopping board● Large plastic funnel● Water bath, set at 60 °C● Ice, in a jug● 250 ml beakers, 2● Coffee filter paper (do not use laboratory

filter paper)● Onion, about the size of a cricket ball● Washing-up liquid, 10 ml (do not use the

thicker, concentrated type)● Table salt, 3 g● Distilled water, 100 ml● 10 ml plastic syringe, for measuring out liquids● Boiling tube● Glass stirring rod● Protease enzyme, e.g.Neutrase (Novo

Nordisk) , 2–3 drops● Ice cold ethanol, about 6 ml (IMS is suitable).

EIBE

This protocol comes from the 'Plant DNA Investigation Kit', which is available from the: National Centre for Biotechnology Education, The University of Reading, PO Box 228, Whiteknights, READING, RG6 6 AJ. The United Kingdom. Tel: + 44 (0) 118 9873743 Fax: + 44 (0) 118 9750140 eMail: [email protected] Further details and protocols can be found at the NCBE Web site: http://www.reading.ac.uk/NCBE


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Isolation ofDNA fromonions

Add the choppedonion to the saltywashing-up liquidsolution.

The washing-up liquidbreaks down the cellmembranes —releasing DNA fromthe nucleus insideeach cell.

10 ml washing-up liquid3 g table salt100 ml water1 medium-sized onion

Add the table salt to thewashing-up liquid. Make upto 100 ml with water. Stirwell to dissolve the salt.

Stand the beaker inhot water at 60 °C for15 minutes.

Hot conditions speedup the process ...

... and denatureDNases that mightdegrade the DNA.

Cool the mixture bystanding the beaker ina jug of ice for a fewminutes.

... but leave it too longand the DNA getsbroken up too ... so anice bath is necessary.

Filter the blendedmixture.

This separates thecell wall materialfrom the DNA andproteins, which arenow in solution.

Blend for no morethan 5 seconds.

The blender helps tobreak open the onioncells — but do notblend for too long oryou will shear the DNA.

Add 2–3 drops ofprotease enzyme toabout 10 ml of theonion extract.

The protease breaksdown the protein inthe solution. Only alittle is needed.

Very carefully pour anequal volume of ice coldethanol onto the surfaceof the onion extract.

The ethanol* MUST be icecold — keep it in the freezerovernight beforehand.

* you can use industrial methylated spirits (IMS)

DNA forms in the upper(ethanol) layer.

DNA does not dissolve inethanol — so it comesout of solution into theupper layer.

1 2 3 4

765 8

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Amylaseproduction bysoil microbes

Aim● To screen naturally-occurring soil

microorganisms for amylase production.

Prerequisite knowledgeStudents will need to understand standardmicrobiological procedures, including aseptictechniques. Knowledge of the starch-iodinereaction would be helpful.

Advance preparationIodine solution should be prepared at least aday ahead. The iodine crystals take a while todissolve completely.Starch/nutrient agar and bottles of sterile

water should be prepared before the lesson.Ideally, soil samples should be air-driedbefore use.Sterile cotton wool swabs. The plastic shaftof shop-bought cotton wool swabs will meltwhen they are autoclaved. Some commercialcotton swabs may also be treated with an anti-microbial agent. Make your own swabs forthis investigation by twirling a small amountof cotton wool round the tip of a cocktailstick. Autoclave in a McCartney bottle orloosely-wrapped in aluminium foil at 121 °Cfor 15 minutes.

OrganisationPreparation and inoculation of Petri dishes:

45 minutesObservation of results, 2–3 days later:

15 minutes

Equipment and materialsRequired by each student or group of students(It is assumed that normal laboratoryequipment is also available)

● Sterile Petri dish, containing 15–20 ml ofsterile starch/nutrient agar, preparedfrom commercial nutrient agar with 0.2%soluble starch added

● 1 g of dry soil, taken from the surface 10 cm● A solution of iodine, made by dissolving

1 g of iodine crystals and 2 g ofpotassium iodide in 300 ml of distilledwater.

● 15 ml of sterile distilled water, in aMcCartney bottle

● Sterile, home-made cotton wool swab(see Advance preparation, above).

● Marker pen (to label Petri dish)

Starch is made from glucose units linked toform either a linear polymer called amyloseor a branched polymer called amylopectin.These polymers are broken down by extra-cellular amylases that are produced by manykinds of organisms, including bacteria andfungi. Large numbers of microbes arescreened to find those suitable forcommercial enzyme production. In thisinvestigation, soil microbes are screenedfor extra-cellular enzyme production.

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Glucose units in amylose and amylopectin are linked byα−(1→4) bonds; in amylopectin, side branches arelinked to these chains by α−(1→6) bonds. The majoramylases that are available commercially are:

α-amylase – hydrolyses α−(1→4) bonds in glucosepolymers, but only within chains, yielding shorterchains (dextrins). Obtained commercially frombacteria (e.g. Bacillus spp.).

β-amylase – hydrolyses α−(1→4) bonds in glucosepolymers, breaking off successive maltose unitsfrom the ends of the chains. Cannot bypass α−(1→6) bonds. Obtained commercially from barleyand malt.

amyloglucosidase – breaks α−(1→4) bonds, cleavingglucose units progressively from the ends of thechains. Also hydrolyses α−(1→6) bonds, but onlyslowly. Obtained commercially from the fungiAspergillus spp. and Rhizopus oryzae.

pullulanase – hydrolyses α−(1→6) bonds. Obtainedcommercially from the bacteria Bacillusacidopullulyticus and Klebsiella pneumoniae.

α-amylase

β-amylaseamyloglucosidase(glucoamylase)

pullulanase

Sites of enzymeaction on amylopectin

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★


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colour of the iodine solution) develop alongthe edges of the colonies if themicroorganisms have degraded starch.

Safety precautionsIMPORTANT: In some countries, it may beforbidden to carry out this investigation, as plates,inoculated with unspecified organisms, are openedafter incubation. If necessary, a culture of Bacillussubtilis could be used instead of soil organisms.Standard microbiology safety procedures shouldbe followed when carrying out this work and whendisposing of cultures, although aseptic techniquesare not strictly necessary up to the inoculation ofthe plates. It is not advisable to subculture(unidentified) organisms from the inoculatedplates. Iodine is toxic, and must be handled with care.

Procedure1. Place 1 g of dry soil in 15 ml of sterile distilled

water. Agitate well to disperse the soil.2. Inoculate the starch/nutrient agar plate, using

a sterile cotton wool swab to streak soilsuspension over the surface of the agar.

3. Label the Petri dish with your initials, the dateand the source of the inoculum.

4. Incubate the inoculated plate, inverted, for2–3 days at 30 °C.

5. Carefully pour iodine solution into theincubated plates until the entire surface of theagar is covered to a depth of about 1 mm.Spots where starch is still present are tintedblue-black (iodine molecules enter the centralspace of the helical chains of glucosemolecules in starch). Light brown zones (the

StarchNutrient

agaraaaaaaaaIncubate the plateat 30°C for 2–3 days

Suspend 1 g of soilin 15 ml of sterilewater

Prepare a Petri dish ofsterile starch/nutrient agar

Inoculate the agar mediumwith soil suspension, usinga sterile cotton wool swab

Stain the plate with iodinesolution, to show where starchhas been broken down

Amylase productionby soil microbes

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productionor a pipette filler and 2 sterile, plugged pipettes

● Discard beaker containing 5% solution ofChlorate I solution e.g. Domestos (Lever)

● Congo red solution (made with 1 mg perml of water)

● 1M sodium chloride solution● Ethanol (for flaming cork borer)● Cork borer, 5 mm diameter● Incubator set at 25–30 °C● Pen for marking Petri dishes

Procedure

1. Dip a 5 mm diameter cork borer into alcohol.Set fire to the alcohol and let it burn away.CAUTION! Take care to hold the borerhorizontally while doing this so that flames donot travel up the centre of the borer and burnyour hand.

2. Hold the lid of a CMC medium plate slightlyto one side and use the borer to cut a well inthe agar. Remove the agar plug from theborer if necessary using a mounted needle.

3. Repeat steps 1 and 2 so that you have twowells in the agar.

4. Label each well on the base of the Petri dish.A suitable code would be: C (Cellulomonas); W(sterile water, a ‘control’).

5. Into the appropriate well place 0.2 ml of eithermicrobial culture or sterile water, using aseparate sterile syringe or pipette for each.Place the syringes, as they are used, in a beakerof disinfectant.

6. Incubate the plates for up to a week at 25–30 °C. Cellulomonas will produce clear zones upto 16 mm in diameter after 48 hours at 30 °C.

After incubation...7. Flood the plates with Congo red solution for

15 minutes, then de-stain with the salt solutionfor 10–15 minutes. Unstained areas indicatewhere the CMC has been broken down to β-(1→4) glucans that contain seven or fewerglucose residues. The diameter of the clearzone can be measured to provide aquantitative comparison of cellulolytic activity.

Extension1. Soil suspensions can be pipetted into wells on

the plates to screen their microbial flora forcellulase activity.

2. The same technique can be used to assay theactivity of commercial cellulase preparations.

3. The course of cellulase breakdown can befollowed over several days by using duplicateplates.

Aim● To provide a quantitative assay of cellulase

production by Cellulomonas.

Advance preparationCultures of Cellulomonas in nutrient broth (e.g.in McCartney bottles) should be prepared forclassroom use. This should be done two or threedays before it is intended to carry out thispractical work. The cultures should be incubatedat 25–30 °C.

CMC medium contains the following: 0.5 g carboxymethylcellulose (a soluble form ofcellulose); 0.1 g NaNO

3; 0.1 g K

2HPO

4; 0.1 g

KCl; 0.05 g MgSO4; 0.05 g yeast extract; and 0.1 g

glucose in 100 ml of water. The medium shouldbe solidified using 1.7 % w/v agar.

OrganisationPreparation and inoculation of Petri dishes:

45 minutesObservation of results, 2–3 days later:

15 minutes


● Slope culture of Cellulomonas sp.● Sterile Petri dish, containing 15 ml of

sterile CMC medium (see Advancepreparation, above)

● Sterile water (dispensed into a McCartneybottle)

● Sterile 1 ml syringes (without needles), 2

There is great interest in utilisingcellulose wastes as feedstocks forfermentation processes, therebyconverting low cost starting materialsinto products of greater value. Mostcommercial cellulases are produced bysubmerged fermentation of the fungusTrichoderma reesei. However, thebacterium Cellulomonas grows morerapidly on a Petri dish and theproduction of extra-cellular cellulasesby it is easy to measure.

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2. Cut and remove two plugsof agar from the plate,forming two 'wells'

1. Sterilize acork borerby flamingit withethanol

3. Inoculate one well withCellulomonas culture

Put sterile water into the second well

4. Incubatethe plateat 30 °Cfor 48hours

5. Stain the plate

Measure the diameter of the area inwhich cellulose has been degraded

IMPORTANT: If soil samples are screenedfor cellulase-producers (Extension, Activity 1),local regulations may forbid the reopening ofinoculated plates.

Safety precautionsStandard microbiological safety procedures,including aseptic techniques, must beobserved when carrying out this work.

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Advance preparationActive cultures of Streptomyces griseus arerequired. These must be grown in for 2–3days on plates, each containing 15–20 ml ofbasal agar medium.

A culture with which to inoculate theseplates must also be prepared in advance. Todo this, resuspend a loop of Streptomycesfrom a slope culture in 1 ml of sterile basalbroth, then pipette this into a test tube intowhich 5 ml sterile basal broth has been placed.Incubate the culture for 24 hours at 30 °C.

Inoculate the Petri dishes with the overnightculture of Streptomyces griseus by streaking theplate with a single vertical line as far over tothe left as possible so that the right part of theculture medium remains sterile.

Incubate the plates for 3–4 days at 30 °C.

In addition, prepare overnight cultures oftest organisms (chosen from Bacillusmycoides, Candida utilis, Escherichia coli K-12,Micrococcus luteus, Pseudomonas fluorescens).

OrganisationPreparation of the media and inoculua:

60 minutes +Initial incubation of the Streptomyces culture:

24 hours, then an additional 72–96 hoursInoculation of plates: 20 minutesIncubation: 24–72 hours


● Access to an incubator, set at 30 °C● Inoculating loop● Sterile Petri dish, containing 15–20 ml of

basal agar medium, onto whichStreptomyces griseus has been streaked andallowed to grow for 48–72 hours (seeAdvance preparation, above)

● A selection of the following organisms, onagar slopes, as test organisms*:- Candida utilis (a yeast)- Micrococcus luteus (a bacterium)- Pseudomonas fluorescens (a bacterium)- Bacillus mycoides (a bacterium)- Escherichia coli K-12 (a bacterium)

*Refer to local regulations for information regardingthe use of these organisms in schools (the use of somemay be prohibited in certain countries).

Antibioticproduction

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Many microorganisms produce

antibiotics — substances which inhibit

the growth of or kill their bacterial

competitors. Since the development of

penicillin in the 1940s (produced by the

fungus Penicillium), these substances have

been highly successful in the fight against

disease. Today, the most important

antibiotics are produced by the bacterium

Streptomyces. More information about the

action of antibiotics, and the

phenomenon of bacterial resistance is

provided in the accompanying pages.

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Below: a three-day-old culture of Streptomycesgriseus (left). The test organisms are (from thetop): Candida utilis, Micrococcus luteus,Pseudomonas fluorescens, Bacillus mycoides,Escherichia coli.

Aim● To demonstrate the production of the

antibiotic streptomycin by Streptomycesgriseus, and to show its effect on thegrowth of a variety of microorganisms.

Prerequisite knowledgeOrigin and effects of antibiotics; thedevelopment and spread of resistance toantibiotics. (See accompanying text).


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Procedure1. Inoculate each prepared Streptomyces plate

with the test organisms as follows:a) Sterilise an inoculation loop by passing

it through a Bunsen burner flame andallowing it to cool briefly.

b) Load the sterile loop with culture ofthe test organisms, then make a singlehorizontal streak for each organismon the clear part of the medium (tothe right of the Streptomyces). Thestreaks must always be at right anglesto the Streptomyces culture and bedrawn close up to the edge of it.IMPORTANT! Do not touch the

Streptomyces culture with the loop.

c) Re-sterilise the inoculation loop bypassing it through a flame once more.

Repeat (a)–(c) for each of the testcultures.

2. Incubate the plates for 2 days at 30 °C.3. Examine the plates.

Safety precautionsThis work must be carried out in a laboratory.Standard microbiology safety procedures shouldbe followed when carrying out this work andwhen disposing of cultures. The amount ofantibiotic produced during this investigationdoes not constitute a safety hazard.

NoteStreptomyces griseus produces the antibioticstreptomycin which diffuses into the culturemedium. Streptomycin and allied antibioticsinterfere with protein synthesis by bindingto the 30S component of bacterialribosomes. Some organisms (Bacillusmycoides, Escherichia coli, Micrococcus luteus) aresensitive; others (Pseudomonas fluorescens) areresistant. Yeasts (Candida utilis) are notaffected by streptomycin as they areeucaryotes with a different ribosomalstructure. For more information, refer tothe accompanying text, which describesantibiotic production and action in detail.

Sterilize a wireinoculation loop

Prepare a Petri dish cultureof Streptomyces griseus 3–4days in advance

Streak one of the testcultures onto theStreptomyces plate

Repeat with each of the test cultures.Remember to sterilize the loopbetween each application.

Incubate the plate, inverted,for 2–3 days at 30 °C.

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Making breaddough

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★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Bread-making is one of the oldestexamples of biotechnology, with accountsof leavened bread dating from ancientEgypt (4,000 BC). In the UnitedKingdom, bread is traditionally madefrom a dough of wheat flour, water, saltand possibly fat, depending upon therecipe. This forms a matrix in which yeastis trapped. Amylases in the moistenedflour convert starch to glucose, whichnourishes the immobilised yeast cells.

In addition, the yeast requires a source ofnitrogen. Peptones and amino acids areprovided by partial hydrolysis of flourprotein (collectively termed gluten). Theyeast’s anaerobic respiration generatescarbon dioxide and alcohol.

Gluten contributes to the elasticity andplasticity of the dough, ensuring that thecarbon dioxide remains trapped as itenlarges the air bubbles within the dough,

causing it to rise.

Aims● This outline protocol may be used to

investigate the effect of various recipecomponents which modify eitherflour proteins or enzyme activity.

OrganisationThis activity can be completed in 50minutes, depending upon the conditions(temperature, etc.).

Equipment and materialsFor each batch of dough

● Dried yeast, 1 g● Strong flour, 75 g● Water, 50 ml● Additional ingredients as desired

e.g. ascorbic acid, α-amylase, potassiumbromate (see Extension, below)

● Small beakers for mixing dough in● Stout glass rods for mixing the dough● 100 ml measuring cylinders, 2● Stop clock

Procedure1. Resuscitate the dried yeast in the water.

Add the flour and mix well.2. Roll the dough into a sausage shape, and

place it in one of the measuring cylinders.3. Record the height of the dough in the

cylinders every 10 minutes over a onehour period.

Extension1. What effect do different types of dried

yeast have on the rate at which doughrises? (e.g. normal dried yeast vs. the newer‘mix-in’ type of yeast.)

2. What differences are there between therates at which doughs made with differenttypes of flour rise? (e.g. strong white,white and wholemeal flours. ‘Strong’flours contain plenty of gluten but verylittle α-amylase. Wholemeal flour is rich inα-amylase.)

3. What effect does the flour improverascorbic acid (vitamin C) have on the rateat which the dough rises? Ascorbic acidinteracts with enzymes in the dough tolimit the extent to which sulphydryl bondsare formed between gluten proteins. (Add1 g to the recipe given for dough above.)

4. What effect does the addition of α-amylase have on the rate at which doughrises? How can any observed differencesbe explained?

5. Flour improver potassium bromatefacilitates the formation of sulphydrylbonds between adjacent gluten proteinsso that more elastic, expandable doughsare made. Of course, too much expansionwould weaken the dough. Comparedoughs made using flour with and withoutthis additive.

6. Salt inhibits the activity of proteases, andso prevents gluten from being weakenedinto a sticky mass that can not retaincarbon dioxide gas. Excess salt formsstrong ionic bonds with side chains ofprotein molecules, making them lessstretchy and leading to a tough bread.Excess salt also inhibits yeast growth. Tryto determine the optimum salt content ofdough.


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BASIC DOUGH RECIPE

1. Put 1 g of dried yeast into50 ml of water.

2. Allow the yeast torehydrate, then add 75 g offlour.

3. Mix well.

1. Place the dough in a measuringcylinder.

2. Record the volume of the dough at10-minute intervals.

QuestionsTo what extent does the volume ofthe dough reflect the growth rate ofthe yeast? How could you test yoursuggestions?

What other factors (apart from theyeast's activity) might affect thevolume of the dough?

Additional information

On food and cooking. The science and lore of thekitchen by Harold McGee (1991) HarperCollins Publishers. ISBN: 0 00 412657 2.

Pritchard, P.E. (1992) ‘Studies on thebread-improving mechanism of fungalalpha-amylase’Journal of Biological Education 26, (1) 12–18.

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Yeast cells immobilised in a matrix ofcalcium alginate.

Ordinary bakers’ yeast, Saccharomyces

cerevisiae, is unable to ferment the sugarlactose. The enzyme β-galactosidasebreaks down lactose to glucose andgalactose. Yeast that is co-immobilisedwith this enzyme is able to grow in amedium that contains lactose. Of the twosugars formed by enzyme action, glucoseis used preferentially. Once supplies ofthis sugar have been exhausted, the yeastadjusts its metabolism and the otherbreakdown product of lactose, galactose,is utilised. The activity of the yeast isreadily-monitored simply by measuringthe volume of gas (carbon dioxide)evolved during the fermentation.

Aim● To provide an introduction to the

quantitative study of fermentation

Advance preparationSodium alginate is not readily soluble in water.Both hot water and stirring are necessary todissolve it. Sodium alginate solution musttherefore be made up in advance. If you wishto store sodium alginate solution, it isadvisable to autoclave it first. Important: usedistilled or dionised water for all solutions, as calciumions in tap water will cause the alginate to ‘set’.

OrganisationImmobilised yeast cells can be prepared in 10–15minutes. Fermentation can take up to a week.


● 4% sodium alginate solution, 10–15 ml● 1.5% calcium chloride solution, 100 ml● 10 ml syringe, without needle● Tea strainer● 200 ml beakers, 2● 250 ml conical flasks, 2● Bungs, to fit fermentation flasks, bored

and fitted with delivery tubes, 2● Large e.g. 500 ml beakers, 2● 100 ml measuring cylinders, 2● Chemical sterilizing solution e.g. sodium

metabisulphite solution or a proprietary

Photograph courtesy D

r Duncan C

asson

product such as Milton (Sodiumhypochlorite solution, Procter & Gamble).

● 13% sodium chloride solution, about 1litre (ordinary table salt is adequate forthis work)

● 100 ml of medium, containing 2 g glucose,1 g yeast extract and 1 g of peptone

● 100 ml of medium, containing 2 g lactose,1 g yeast extract and 1 g of peptone

● β-galactosidase enzyme, e.g. Lactozym(Novo Nordisk), 2 ml

● Bakers’ yeast, fresh or dried

ProcedurePrepare the immobilised enzyme and yeast

pellets as follows:

1. Mix the dried yeast with 25 ml distilledwater in a small beaker. Cover and leave torehydrate for 10 minutes at roomtemperature.

2. Mix the sodium alginate solution and β-galactosidase together in the other smallbeaker. Stir in 10 ml of the yeastsuspension.

3. Draw up some of the yeast / enzyme /alginate mixture into the syringe. Add it, a


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drop at a time, to the calcium chloridesolution in the second large beaker.

4. Leave the immobilised enzyme / yeast cellpellets to harden in the calcium chloridesolution for 10 minutes.

5. Separate the pellets from the solutionusing the tea strainer. Rinse them well withtap water.

The pellets may be stored in sterile water in arefrigerator for up to 3 days before they are used.

Set up the fermentation vessels as follows:

1. Sterilize the flasks using sodiummetabisulphate solution or a similarchemical. Rinse the flasks with water aftersterilization is complete.

2. Add the sterile growth media to the flasks.Use glucose medium for one (as acontrol), and lactose medium in the other.

3. Add an equal number or mass ofimmobilised yeast pellets to each flask.

4. Stopper the flasks with bungs that havebeen fitted with delivery tubes.

5. Maintain the flasks at 21–25 °C. Collectthe gas that is produced over a 13%solution of sodium chloride (carbondioxide will not dissolve in this solution).Record the volume of gas that has beencollected at convenient intervals.

6. Plot the results on a graph, showing thevolume of gas evolved against time.

Extension1. Different types of yeast e.g. wine-makers’

or bakers’ yeast may be used.2. The concentration of β-galactosidase,

incubation temperature and othervariables may be altered.

Safety precautionsBuild-up of gas within glass vessels could bedangerous. Ensure that the fermenter vesselsare adequately vented. Care should be takenwhen using chemical sterilizing agents, andany guidelines for their use given bymanufacturers must be followed.

Ca2+

The cells or enzymes to be entrapped are firstmixed with a solution of sodium alginate. Thisis then dripped into a solution containingmultivalent cations (usually Ca2+). Thedroplets form spheres automatically as theyfall, entrapping the cells in a three-dimensionallattice of ionically cross-linked alginate (seediagram, above).

For more information, see Smidsrød, O. andSkjåk-Bræk, G. (1990) Alginate as animmobilization matrix for cells. Trends inBiotechnology 8 (3) 71–78.

Entrapment within calcium alginate is themost widely used technique for immobilisingcells. It is especially suited to living cells as itrequires only very mild conditions.Applications of this versatile method includeimmobilisation of living or dead cells inbioreactors, entrapment of plant protoplastsand plant embryos (‘artificial seeds’) formicropropagation, immobilisation ofhybridoma cells for the production ofmonoclonal antibodies, and the entrapment ofenzymes and drugs (see table, overleaf).

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Yeast cells thrive on the sugarsformed by the enzyme, making

carbon dioxide and alcohol

Yeast, lactase enzyme and sodium alginate solution

Add a dropat a time

Enzyme inside the beadsbreaks down lactose toglucose and galactose

®

The University of Reading, United Kingdom

NATIONAL CENTRE FOR BIOTECHNOLOGY EDUCATIO

N

Enzymes for Schools and Colleges

Novo Nordisk Lactozymβ-galactosidase from Kluyveromyces lactis

100 ml

Store at 4°C. Do not freeze.

Saccharomyces cerevisiaeFermentation results

Plot the volume of gasevolved against time

Rinse withwater

Examples of uses of alginate-immobilised cells (after Smidsrød and Skjåk-Bræk, 1990)

Cells Product/purpose

BacteriaErwinia rhapontici IsomaltulosePseudomonas denitrificans Cleaning of drinking waterZymomonas mobilis Ethanol

CyanobacteriaAnabena sp. Ammonia

FungiKluyveromyces bulgaricus Hydrolysis of lactoseSaccharomyces cerevisiae EthanolSaccharomyces bajanus Champagne production

Cells Product/purpose

AlgaeBotryococcus braunii Hydrocarbons

Plant cellsChatharanthus roseus Alkaloids for cancer therapyVarious plants Artificial seedsPlant protoplasts Cell handling, microscopy

Mammalian cellsHybridomas Monoclonal antibodiesIslets of Langerhans Insulin/implantationFibroblasts or lymphoma cells Interferons (α or β)


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The microbialfuel cell

How to assemble amicrobial fuel cell(the exact dimensions areunimportant — the one shown isroughly 55 mm x 55 mm)

cationexchangemembrane

chamber glued to endplate with aquarium

cement

J-Cloth preventselectrode from touching

membrane

neoprenegasket

hole through whichchamber is filled

terminal ofcarbon fibreelectrodeaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Advance preparationSolutions of reagents should be prepared inadvance. Pre-soak the cation exchangemembrane in distilled water for 24 hoursbefore use. The dried yeast can beresuscitated as the fuel cell is assembled.

OrganisationAssembly (through to electricity generation)takes about 30 minutes.


● Perspex (ICI) fuel cell, cut from 4 mmthick sheet

● Neoprene gaskets, 2● Cation exchange membrane, cut to fit

between chambers of the fuel cell. Themembrane may be re-used indefinitely, but willmelt if it is autoclaved.

● Carbon fibre tissue electrodes, cut to fit, 2● J-Cloth (Johnson & Johnson), cut to fit

inside cell, 2 pieces● 10 ml plastic syringes, 2, for dispensing liquids● Petri dish base or lid on which to stand

fuel cell● Electrical leads with crocodile clips, 2

Aims● To provide a stimulating introduction to

the study of respiration● To permit the study of some of the factors

which influence microbial respiration● To show how waste organic material may

be used to generate electricity

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For decades, microbes that produceelectricity were a biological curiosity.Now, researchers foresee a use for themin watches and cameras, as powersources for the Third World and forbioreactors to turn industrial waste intoelectricity. The microbial fuel celldescribed here generates a smallelectrical current by diverting electronsfrom the electron transport chain ofyeast. It may be used to studyrespiration in a novel and stimulatingmanner. Further information is given inBIO/technology Education, Volume 1,Number 4, pages 163–168.

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How the fuel cellworks

● 0–5 V voltmeter or multimeter and / orlow current motor

● Scissors

All the solutions listed below should be made up in

0.1 M phosphate buffer, pH 7.0, instead of water

● Dried yeast, made into a thick slurry in0.1M phosphate buffer (do not addglucose solution without first resuscitatingthe yeast in buffer)

● 10 mM methylene blue solution, 5 ml● 1 M glucose solution, 5 ml● 0.02 M potassium hexacyanoferrate (III)

solution, 10 ml (also called potassiumferricyanide)

To make 0.1 M phosphate buffer, pH 7.0

Dissolve 4.08 g Na2HPO

4 and 3.29 g

NaH2PO

4 in 500 ml distilled water.

Procedure1. Cut out two carbon fibre electrodes as

shown on the accompanying page.2. Cut out two pieces of J-Cloth to fit inside

the fuel cell.3. Assemble the fuel cell as shown on the

accompanying page.4. Stand the assembled fuel cell on a Petri

dish base or lid to catch any liquid whichmay leak.

5. Combine the yeast slurry, glucose andmethylene blue solutions. Syringe themixture into one chamber of the fuel cell.

6. Syringe potassium hexacyanoferrate (III)solution into the other side of the cell.

7. Connect a voltmeter or multimeter (viathe crocodile clips) to the electrodeterminals. Fuel cells of this type typically

generate between 0.4–0.6 V. A currentshould be produced immediately — if the meterregisters zero, check the connections and ensurethat the carbon fibre electrodes are not touchingthe cation exchange membrane.

Extension1. Several fuel cells may be joined together

to give a greater voltage. Increasing thesize of the cell (or the electrode area) willincrease the current generated (but notthe voltage).

2. Different mediators and / or types ofyeast e.g. wine-makers’ or bakers’ yeastmay be used. Note: For safety reasons, the useof this fuel cell with other microorganisms is notrecommended.

3. Investigate the effect of temperature onthe action of the fuel cell (remember toconsider what ‘controls’ are necessarywhen making comparisons of this type).

Safety precautionsPotassium hexacyanoferrate(III) is poisonous. Eyeprotection should be wornwhen handling this material. Ifthe solution comes into contact

with the eyes, flood them with water and seekmedical attention. If swallowed, give plenty ofwater to drink and seek medical attention. Localregulations should be observed when disposingof used solution.

AcknowledgementThe microbial fuel cell was developed by DrPeter Bennetto at King’s College, London.

indicates electrons

methylene blue(reduced)

methylene blue(oxidised)

V

Yeast cellselectron transport chain

Glucose

Carbondioxide 4 H+

4 4

Anode Cathode

Fe(CN)64-

3-Fe(CN)6

Assembled fuel cell

potassiumhexacyanoferrate

cation exchangemembrane


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RNatural genetransfer bybacterialconjugation

Three types of natural gene transfer arerecognised:

Transformation is the uptake of free DNAfrom the cell’s surroundings;

Transduction is the movement of DNAfrom one cell to another through theintervention of a bacteriophage;

Conjugation is the transfer of specialised‘F-plasmids’ through a narrow tube (sexpilus) which links two bacterial cells (seeFigure 1).

All of these mechanisms (transformationespecially) are used by genetic engineersto ferry selected genes into bacteria.

In the following investigation,

conjugation between two naturally-

occurring bacterial strains is examined.

Note: This work uses naturally-occurring

bacteria, plasmids and processes and is not,

therefore, ‘genetic engineering’.

There are various natural methods bywhich genes may transferred betweenbacteria. These mechanisms haveprobably evolved to allow organisms toadapt to rapidly-changing environmentalconditions. As a rule, genes with thegreatest mobility are located on plasmids.

Plasmids are rings of DNA whichreplicate independently of the bacterialchromosome. They carry a small numberof genes conferring on their holders suchthings as an ability to break down harmfulsubstances in the environment, like heavymetals or antibiotics.

Donor

Recipient

F-plasmid

1. F-plasmid unwoundand replicated

2. Single-stranded DNApasses into recipientcell through pilus

3. ComplementaryDNA synthesised,forming new F-plasmid

Figure 1: Conjugation and transfer ofF-plasmids between bacterial cells.

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Figure 3:How donor, recipientand transconjugantstrains aredistinguished onMacConkey agarthat containsstreptomycin

F-plasmids

The so-called ‘F-plasmids’ (F stands for‘fertility’) contain genes which enable thetransfer of a copy of the plasmid betweendonor and recipient cells (in other words,bacterial conjugation).

F-plasmids may carry other genes as well.For example, in the investigation describedhere, the donor strain carries a plasmidcalled F Lac. It is so named because it carriesgenes which allow its bacterial host tometabolise lactose (the bacterium istherefore called a Lac+ strain). In contrast,the recipient strain, with no plasmid, isunable to metabolise lactose (it is a Lac -

strain).

On plates of MacConkey agar these differentstrains can be distinguished by theirappearance: the donor strain forms redcolonies, while the recipient strain formswhite colonies (see Figure 2).

When donor and recipient strains are mixed,F Lac plasmids are transferred from thedonor to the recipient. In this way therecipient acquires the ability to metaboliselactose.

A genetic ‘trick’ enables the donor,recipient and transconjugant strains tobe distinguished. A recipient strain ischosen with a chromosomal gene thatrenders it insensitive to the antibioticstreptomycin. The donor strain has nosuch gene and is inhibited bystreptomycin. Hence the recipientstrain and transconjugants can beidentified by their ability to grow on amedium that contains streptomycin (Figure 3).

RecipientEscherichia coli

(Lac - strain)

DonorEscherichia coli

(Lac+ strain)

Small whitecolonies

Redcolonies

Lac+ gene onplasmid

Figure 2:The appearance of donor and recipient strainson ordinary MacConkey agar

RecipientEscherichia coli

K-12 CSH36

DonorEscherichia coli

K-12 L17

Small whitecolonies

Redcolonies

Lac+ gene onplasmid

Transconjugant

Nocolonies

Streptomycin resistancegene on chromosome

Lac+ gene onplasmid

Streptomycin resistancegene on chromosome


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Aims● To provide a stimulating introduction to

bacterial genetics● To provide an opportunity for students to

discuss issues associated with natural genetransfer e.g. the spread of antibioticresistance and ‘risk assessment in genetechnology

Advance preparationThe following media should be prepared andsterilised:

● 3 small (e.g. 100 ml) conical flasks, eachcontaining 10 ml of sterile nutrient broth;

● Sterile MacConkey agar with addedStreptomycin sulphate [200 mg per ml].After the agar has cooled to 50 °C (handhot) it should be divided between sterilePetri dishes (allow 15–20 ml per dish).

● The cultures used are:Donor strain:

Escherichia coli K-12 CSH36(DSM Number 6253);

Recipient strain:

Escherichia coli K-12 L17(DSM Number 6254).

These naturally-occurring strains come fromthe German Collection of Microorganismsand Cell Cultures (DSM). Both cultures aresupplied freeze-dried, in glass ampoules. Theampoules must be opened in the correctmanner to ensure that their contents are notcontaminated and that the user is not exposedto unnecessary hazards (see accompanyinginstructions).The donor strain is difficult tomaintain on a slope, so a freshly-resuscitatedculture is necessary.

Overnight cultures of the donor andrecipient strains should be prepared asfollows:

1. Inoculate one flask of sterile nutrientbroth with the donor strain (labelled‘Donor’), and another with the recipient(labelled ‘Recipient’);

2. Incubate both flasks overnight at 37 °C,ideally in a shaking water bath.

‘Matings’ of these two cultures areperformed as follows:

1. Using a sterile pipette, aseptically add 0.8 mlof the donor strain to the third conical flask,containing 10 ml of sterile nutrient broth.

2. Using a sterile pipette, aseptically add 0.2ml of the recipient strain to the sameconical flask.

3. Label the flask appropriately and incubateit at 30 °C for 16–24 hours. Note: shake theflask only VERY GENTLY duringincubation — vigorous action will break the piliwhich link conjugants.

Sterile cotton wool swabs are prepared bytwirling a small amount of cotton wool roundthe tip of a cocktail stick. Autoclave these in aMcCartney bottle or loosely-wrapped inaluminium foil at 121 °C for 15 minutes.

OrganisationPreparation of media: 60 minutesInitial incubation: 48 hoursInoculation of plates: 15 minutesIncubation: 24 hoursObservation of results: 20 minutes


● Access to an incubator, set at 30 °C● Sterile Petri dish, containing 15–20 ml of

MacConkey agar, with added Streptomycin● The following cultures, grown in advance on

nutrient broth:- Donor strain- Recipient strain- Mixed mating of donor and recipient strains

● 3 sterile, home-made cotton wool swabs● Small beaker, containing disinfectant

solution e.g. 3% Domestos (Lever) solution, inwhich to place used swabs

● Marker pen (to label Petri dish)

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...


Procedure1. Draw three lines on the base of one of the

Streptomycin / MacConkey agar plates,dividing it into three equal segments.

2. In the middle of each segment, draw acircle about 10 mm in diameter. Label one‘D’ (for the donor strain), one ‘R’ (for therecipient strain) and the final circle ‘M’ (formixture of the two strains — formingtransconjugants).

3. Use a sterile cotton swab to inoculate eachmarked area on the plate with theappropriate bacterial strains. Remember touse a fresh swab for each culture. Disposeof used swabs into disinfectant.

4. Allow the plates to stand for a fewminutes, until liquid can no longer be seenon the surface of the agar medium.Incubate the plates, inverted, for one totwo days at 30 °C.

ExtensionSeveral quantitative versions of this work maybe carried out, for example:1. By diluting the donor and recipient

cultures in sterile Ringer’s or 10-3 MgSO4

solution, the optimum ratio of donor torecipient may be determined.

2. The optimum mating time required forconjugation may be determined.

Safety precautionsThis work must be done in a laboratory.Standard microbiology safety procedures,including aseptic technique, should befollowed when carrying out this work andwhen disposing of cultures.

AcknowledgementsThis investigation is a simplified version ofone devised by Professor Patricia Nevers ofthe University of Hamburg. In turn, ProfessorNevers’s protocol was based on that of E.Härle and R. Hausmann at the University ofFreiburg.


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CAUTION!Safety glasses must be worn,as glass splinters may scatterwhen the ampoule is opened.

Opening an ampouleA

PPE

ND

IXResuscitating a freeze-dried culture

1. With forceps, remove the cotton woolplug and briefly flame the opening of theinner glass tube.

2. Aseptically add about 1 ml of sterilenutrient broth to the contents of theinner tube.

3. Reflame the opening of the tube andreplace the cotton wool plug. Leave thetube for 20 minutes to allow the driedculture to revive.

4. Using a flamed inoculation loop, mix thecontents of the tube well and pour theminto a sterile test tube containing about 5ml of nutrient broth solution.

5. Incubate the culture at 30 °C overnight.

The next day...

6. Use a flamed inoculation loop to streak adrop of the prepared suspension ontothe surface of a nutrient agar plate. Thisis done to check whether the culture has beencontaminated—only one type of colony shouldgrow on the plate.

1. Heat the pointed tip of the ampoule in aBunsen burner flame. Rotate the ampouleas you heat it (see diagram).

2. With a pipette, drop, at most, two or threedrops of cold water onto the heated tip ofthe ampoule. The glass should shatter.

3. Tap the shattered tip of the ampoule firmlyyet carefully with a pair of forceps,catching the shards of glass in a Petri dishbase. Ensure that the broken glass isproperly disposed of.

4. With a pair of forceps, remove the glasswool plug which retains the inner glasstube within the ampoule.

5. Carefully tip the inner tube out into asterile Petri dish, replacing the lid on thedish as you do so.

Openingan ampoule

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GE

NE

TR

AN

SFE

Rare unable to penetrate intact plant cell walls.Sap from damaged cells ‘lures’ nearbybacteria and activates the gene transfer.They multiply in the area around thewound and penetrate the intercellulararea, attaching themselves to the plant’scell walls. The Agrobacterium’s plasmid istransferred and eventually integrated intothe plant’s own chromosome, from whereit directs the production of opines, whichthe Agrobacterium uses as a source ofcarbon and nitrogen.

Aims● In the following investigation Bryophyllum

= Kalanchoe sp. (which is both easy to growand propagate) is infected under variousconditions with the naturally-occurringform of Agrobacterium. Within 4 weeks,tumour growth can be observed. Thefollowing variations in the experimentalprotocol can be investigated:

A. Mode of infection:- scratch a wound and infect immediately

with Agrobacterium;- scratch a wound and coat with

Agrobacterium the following day;- scratch a wound and infect with

Agrobacterium. Cover with a damp pieceof paper tissue;

- scratch a wound but DO NOT infect itwith bacteria;

- apply Agrobacterium to unwounded areasof plants.

B. Site of infection:- stem;- leaf surface;- shoot tip.

Advance preparation1. Propagation of the host plants (students

can do this — at home if appropriate);2. Preparation of nutrient medium and

cultures of Agrobacterium tumefaciens. freshcultures must be prepared at least 2 weeksbefore they are to be used.

OrganisationGrowing the host plants (if necessary):

4 monthsPreparing the Agrobacterium culture:

2 weeksInfecting the plants with Agrobacterium:

20 minutesObserving the tumour growth:

3 to 4 weeks

Plant tumours are a serious nuisance inagriculture, horticulture and viticulture.They often develop after tiny injuriesoccur to plants through soil cultivation,following frost damage or during grafting.

At the turn of the century it was noticedthat certain tumours were alwaysassociated with bacterial infection. Thebacteria involved were namedAgrobacterium tumefaciens (latin: agar =ground; tumour = swelling; facere = to do).Only towards the end of the 1970s waslight finally shed upon the complexinterrelationship between plants andAgrobacterium.

The infective bacteria introduce a smallpiece of their genetic information (aplasmid) into the genome of the plantcells, forcing the infected cells to follow abacteria-friendly programme. This causeseach affected plant cell to divide. Atumour develops which serves as a habitatand provides unusual amino acidderivatives that are required by theuninvited bacterial guests.

The methods of gene transfer used byAgrobacterium have been adapted and areused by plant breeders to transferdesirable genes without time-consumingcross-breeding. As a result, modifiedforms of Agrobacterium have become animportant tool in gene technology.

Agrobacterium is rod-shaped and aboutthe same size as E. coli; 1 to 3 µm inlength. Agrobacterium tumefaciens retainsits virulence in the soil, where it livesaerobically in the upper layers. It is asaphrophyte, although it can utilizeinorganic nitrogen. Agrobacterium invadesonly dicotyledonous plants and can onlyenter and infect the plants throughwounded areas. This is because the bacteria

Natural genetransfer byAgrobacteriumtumefaciens★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★


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Sterilize amounted needle

Scratch the plantwith the sterileneedle

Apply bacteriato the scratch

Flame theinoculationloop beforeand afteruse


● Sterile water (dispensed into a McCartney bottle)● Inoculation needle● Inoculation loop● Binocular microscope● Scissors● Tie-on labels and marker pen● Paper towels● Adhesive tape● Ethanol, for flaming instruments● Culture of Agrobacterium tumefaciens (on

nutrient agar)● Kalanchoe (Bryophyllum) plants, roughly 3 to

4 months old

ProcedureThe plants used in the trial are treated invarious ways (see introductory notes, aboveand the instructions below).

1. Label each plant with the date and whattreatment it has received. If necessary,identify the infected parts of the plants e.g.with tie-on labels.

2. After treatment, place the plants in a well-lit area and keep them damp (but do notoverwater them!).

3. Observe and record the development ofthe tumours during the following 4 to 6weeks. Examine a piece of the tumourtissue under a binocular microscope andcompare it to a piece of tissue from anormal leaf.

How to infect the plants with Agrobacterium

Method 1 (the wounds are infected immediately)1. Dip an inoculation needle into alcohol and then

ignite the alcohol and allow it to burn off. Scratchthe plant’s surface one or more times.

2. Infect the wounds with Agrobacterium from theculture. To do this, flame an inoculation loopand cool it briefly. Lift up a little of the whitishbacteria culture with the loop and spread itover the wound. Reflame the loop.

Method 2 (the plants are infected 24 hours after theyhave been wounded)1. Wound the plant (as in Method 1).2. Spread the wounded area with

Agrobacterium the next day.

Method 3 (the wounds are infected, then covered withdamp paper tissue)1. Wound and infect the plant as described

above (Method 1).2. Cut pieces of paper from towels and dampen

them with sterile tap water. Place the strips onthe wound and fasten them with adhesive tape.Keep the paper damp for two days.

Method 4 (wounds are not infected)1. Make wounds at the same sites as in Method 1, then

on a second site — cover the latter with damppaper. Do not treat the wounds with Agrobacterium.

Method 5 (infection without wounding)1. Do not wound the plant.2. Apply use a wire loop to aseptically apply

Agrobacterium to one or more parts of theunwounded surface.

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Safety precautionsThis work must be done in a laboratory.Standard microbiology safety procedures,including aseptic technique, should befollowed when handling microbial cultures.

IMPORTANT: A. tumefaciens is a serious plantpathogen. Its use in experimental work is strictlycontrolled in some countries and regions.

Those wishing to carry out such work shouldensure that they comply with local regulations.

AcknowledgementsUta Nellen at the Centre for School Biologyand Environmental Education in Hamburgdevised this investigation. EIBE is grateful forher permission to use this material.

Leaf discs cultivated on aselective nutrient medium -only transformed cells thrive

Unwanted genesremoved from

plasmid

Desired geneisolated from adonor organism

Leaf discs floated on asuspension of

Agrobacterium cells

Whole plants, which nowcontain the introduced gene,

are regenerated

Novel plasmid put intoAgrobacteriumtumefaciens

How modified forms of Agrobacterium tumefaciens are used in gene technology


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Microbiological mediaAppendix 1

UN

IT1European Initiative for Biotechnology Education

Nutrient agar medium and nutrient broth should be prepared from commercially-availablesources, following the directions of the manufacturer. They should be autoclaved before usefor 15 minutes at 121 °C. Prepared media may in general be stored at ~4 °C for severalmonths.

Starch agar mediumNutrient agar 20.7 gStarch, soluble 2.0 g

Make up to 1 litre with distilled water then autoclave for 15 minutes at 121 °C.

MacConkey agar with streptomycinMacConkey agar 50.0 gDistilled water 990 ml

After autoclaving for 15 minutes at 121 °C , allow to cool to 50 °C, then add:

Streptomycin sulphate solution 200 mg/10 ml

These plates must be freshly-prepared. They may be stored for no more than a few days in arefrigerator at ~4 °C.

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Microbiological techniquesAppendix 2

UN

IT1European Initiative for Biotechnology Education

AerosolsAerosols are small microbe-laden dropletswhich may be released into the air byaccident, persist for half an hour or more,and be inhaled. They are the major potentialsource of infection in laboratories. Aerosolsfrom spilt cultures may result in skin and eyeinfections. Ingestion of microbes is also verylikely if cultures are pipetted by mouth.

General laboratory practiceThe following precautions should be taken.

No hand-to-mouth operations should occur(e.g. chewing pencils, licking labels, mouthpipetting). Eating, drinking and smokingmust not be allowed in the laboratory.

It is strongly recommended that studentsshould wear laboratory coats. Any exposedcuts and abrasions should be protected withwaterproof dressings before the practicalwork starts.

Benches should be swabbed with alaboratory disinfectant before and after eachpractical session. Suitable disinfectants areavailable from school science suppliers.

Teachers, technicians and students shouldalways wash their hands after practical work.

Spills and breakagesAccidents involving cultures should be dealtwith as follows:

Disposable gloves should be worn. Thebroken container and/or spilled cultureshould be covered with a cloth soaked indisinfectant. After not less than 10 minutes, itmust be cleared away using paper towels anda dustpan. The contaminated material mustbe placed in an infected waste container ordisposal bag. This must be autoclaved beforedisposal. The dustpan should also beautoclaved or placed in disinfectant for 24hours.

Accidental contamination of skin or

clothing

As soon as possible, anyone who has beensplashed should wash. Severely contaminatedclothing should be placed in disinfectantbefore it is laundered.

Contaminated cleaning cloths should beautoclaved or soaked in disinfectant.

Sources of microbesAll micro-organisms should be regarded aspotentially harmful. The organisms used inthe investigations in this Unit presentminimum risk given goodpractice. They should onlybe obtained fromrecognised suppliers.


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The aims of aseptic techniques are:

a) To obtain and maintain pure cultures ofmicroorganisms;

b) To make working with microorganismssafer.

A ‘pure culture’ contains only one speciesof microorganism,whereas a ‘mixed culture’contains two or more species.

Contamination of cultures is always a threatbecause microbes are found everywhere; onskin, in the air, and on inanimate objects. Toobtain a pure culture, sterile growth mediaand equipment must be used andcontaminants must be excluded. These arethe main principles of aseptic techniques.

It is unrealistic to expect younger students tobe fully accomplished at aseptic techniques.However, some of the activities in this Unitrequire students to transfer culturesaseptically. In such instances, the followingprocedures should be adopted.

Growth media should be sterilized beforeuse by autoclaving. Sterile containers (flasks,Petri dishes, etc.) should be used. Lids mustbe kept on containers to preventcontamination.

Work should be done near a Bunsen burner.Rising air currents from the flame will carryaway any microbes that could contaminategrowth media and pure cultures.

When cultures are transferred, tops and lidsshould not be removed for longer thannecessary. After a lid has been taken from abottle, it should be kept in the hand until it isput back on the bottle. This preventscontamination of the bench and the culture.After removal of the top, the neck of theculture bottle should be flamed for 1–2seconds. This will kill any microbes presentthere and cause convection currents whichwill help to prevent accidental contaminationof the culture from the atmosphere.

With practice, it is possible to hold the bottlein one hand and the wire loop in the other insuch a way that the little finger is free to gripthe bottle top against the lower part of thehand. (In this case, it is important that thebottle top should be loosened slightly beforethe inoculation loop is picked up.) Ifnecessary, two students may work together toperform these operations.

Top heldby finger

Inoculation loops should be heated until theyare red hot along the entire length of the wirepart. This should be done both before andafter transfer of cultures takes place. Theyshould be introduced slowly into the Bunsenburner flame to reduce sputtering and aerosolformation.

When the Bunsen is not in use it should bekept on the yellow flame, so that it can beseen easily. A blue flame about 5 cm highshould be used for sterilizing loops or wiresand flaming the necks of bottles.

Avoid contaminating the work area.Instruments should be sterilizedimmediately after use and used pipettesshould be placed directly into a jar of freshdisinfectant solution.

Aseptic techniques

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...


Label the Petri dish on the base, beforeinoculation. A name, date and the name orsource of the organism used will allow theplate and its contents to be identified.

Where appropriate, use adhesive tape to sealPetri dishes as shown below:

The seal will ensure that the plates are notaccidentally opened or tampered with. Note:do not seal plates completely round the edgesas this could create anaerobic growthconditions within the dish.

BacteriaBacterial cultures in Petri dishes shouldusually be incubated with the baseuppermost, so that any condensation thatforms falls into the lid and not on thecolony. (If there is heavy condensation inthe sterile Petri dish before inoculation, itshould be dried before use.)

After 2–3 days of incubation at 25–30 °C,bacterial colonies will be seen.

FungiPetri dishes containing fungi do not need tobe inverted. Fungal cultures should beincubated for 7 days or so. Room temperature(~21 °C) is sufficient to allow their growth,although an incubator gives greater control.

Glass pipettes should be put in the largerpot. Do not take the pipette filler off theend of the pipette until the tip is in thedisinfectant, otherwise aerosols can becreated. Dirty pipettes should beautoclaved, washed and rinsed before theyare used again.

Contaminated paper towels, cloths andplastic Petri dishes should be put into thedisposable items autoclave bag.

Any contaminated glassware (includingused glass Petri dishes) should be put intothe autoclave bag for glassware.

Glassware that is not contaminated can bewashed normally. Broken glassware shouldbe put in a waste bin reserved exclusivelyfor that purpose. If the glassware iscontaminated it must be autoclaved beforedisposal. Uncontaminated glassware can bedisposed of immediately.

It is very important to dispose of all thematerials used in a practical class properlyto avoid contamination of the laboratoryand people. All containers used for storingand growing cultures must be autoclaved,then washed in disinfectant and rinsed,before re-use.

Two autoclave bags should be available inthe laboratory: one for reusable glasswareand another for disposable materials. Thereshould be a large and a small discard jarnear each work area for pipettes andmicroscope slides. A metal bucket shouldbe available for disposal of any brokenglassware.

Disposable plastic pipettes, microscopeslides and any liquid from cultures shouldbe put into the small disinfectant pot.Plastic pipettes are then autoclaved anddisposed of, microscope slides should besoaked in disinfectant for 24 hours thenwashed and rinsed before re-use.

Incubation of cultures

Disposal and sterilization


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AutoclavingSterilization means the complete destructionof all microorganisms, including their spores.

All equipment should be sterilized beforestarting practical work so that there are nocontaminants. Cultures and contaminatedmaterial should also be sterilized after use forsafe disposal.

Autoclaving is the preferred method ofsterilization for culture media, aqueoussolutions and discarded cultures. The processuses high pressure steam, usually at 121°C.Microbes are more readily killed by moist heatthan dry heat as the steam denatures theirprotein. Autoclaving can be done with adomestic pressure cooker or a purpose-builtautoclave. Domestic pressure cookers can beused in school laboratories but their smallcapacity can be a disadvantage when dealingwith class sets of material.

Principles of autoclaving Two factors are critical to the effectiveness ofthe process. Firstly, all air must be removedfrom the autoclave. This ensures that hightemperature steam comes into contact withthe surfaces to be sterilized: if air is present thetemperature at the same steam pressure islower. The materials to be sterilized should bepacked loosely so that the air can be drivenoff. Screw-capped bottles and jars shouldhave their lids loosened slightly to allow air toescape and to prevent a dangerous build-up ofpressure inside them.

Secondly, sufficient time must be given for heatto penetrate (by conduction) to the centre ofmedia in Petri dishes or other containers. Thetimes for which media or apparatus must be heldat various temperatures for sterilization areshown below:

130120110100

0

200

400

600

800

1000

1200

1400

Temperature (°C)

Ste

riliz

atio

n ti

me

(min

utes

)

Temperature100°C110°C115°C121°C125°C130°C

Holding Time20 hours2.5 hours

50 minutes15 minutes6.5 minutes2.5 minutes

Notice that just a small difference intemperature can result in a great difference inthe time required for sterilization. It is alsoimportant that these temperatures are reachedby all materials to be sterilized for thespecified time e.g. the broth in the very centreof a fermenter vessel.

Hence three factors contribute towards theduration of the autoclaving process:

● penetration timethe time taken for the centre-most part ofthe autoclave’s contents to reach therequired temperature;

● holding timethe minimum time in which, at a giventemperature, all living organisms will bekilled;

● safety timea safety margin; usually half the holdingtime.

Domestic pressure cookers operate at 121°C.Thus the total sterilization time might typically be:penetration time, say 5 minutes; plus 15 minutesholding time; plus a safety margin of 5 or sominutes, giving a total time of 25 minutes.

Vessel Volume Holding timeTest tube 20 ml 12–14 minutesFlask 50 ml 12–14 minutesFlask 200 ml 12–15 minutesFermenter 1 litre 20–25 minutes

CaramelizationPurpose-built autoclaves sometimes operateabove 121 °C, and whilst the savings in timethey offer can seem beneficial, it should beremembered that higher temperatures aredetrimental to certain media. Glucosesolution, for example, is caramelized at hightemperatures, forming compounds whichmay prove toxic to microbes. In the case ofglucose this reaction can be avoided byadjusting the pH of the medium to 4. Aftersterilization the pH can be re-adjusted asnecessary.

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Maillard reactionsA browning reaction (the Maillard reaction)can also be caused by the interaction ofnitrogenous compounds and carbohydrates inthe medium at high temperatures. Here toothe compounds formed are toxic to somemicrobes, so it may in some circumstances benecessary to autoclave the carbohydrate andthe remainder of the medium separately e.g. inthe preparation of milk agar.

Use and routine care of autoclavesThe manufacturer’s instructions shouldalways be followed when using a pressurecooker or an autoclave. Particular careshould be taken to ensure that there isenough water in the autoclave so that itdoes not boil dry during operation. Adomestic pressure cooker requires at least250 ml of water - larger autoclaves mayneed considerably greater volumes. The useof distilled or deionised water in theautoclave will prevent the build-up oflimescale. Autoclaves should be driedcarefully before storage. ‘Pitting’ of theautoclave base may occur if this is not done,seriously weakening the vessel and socausing it to ‘bow’ outwards under pressure.

When the autoclave is used, before the exitvalve is tightened, steam should be allowedto flow freely from the autoclave for about

one minute to drive off all the air inside.After the autoclave cycle is complete,sufficient time must be allowed for thecontents to cool and return to normalatmospheric pressure. The vessel or valvesshould not be opened whilst under pressureas this will cause scalding. Premature releaseof the lid and the subsequent reduction inpressure will cause any liquid inside theautoclave to boil. The agar or broth willfroth up and it may boil over the outside ofthe containers.

Chemical sterilizationMany different chemicals are used forsterilization. The most commonly useddisinfectants in laboratory work are clearphenolics and hypochlorites.

Clear phenolics are effective against bacteriaand fungi but inactive against spores andsome types of virus. They are inactivated tosome extent by contact with rubber, woodand plastic. Laboratory uses include discardjars and disinfection of surfaces.

Hypochlorites (such as bleaches) are notideal for sterilization of used Petri dishesetc. as they can be inactivated by proteinand plastic materials. However, a 5%solution of Domestos (Lever) or Chlorate Isolution is suitable for use in discard jars.

eibe unit 1 : english language version - ipn

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