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it'l i:)'i-:il;I i i i::ji-i-l:; 117 Usingmicroscopy to enhanceundergraduate teaching 127 Letter 136 obituaries i:l l'Tl{,-li:}S 96 Fluorescencemicroscopy Canyou seethe asa researchtool in bacterial light? cellbiology lain Hagan , AgnesCrallert & Jeff Errington SteveBagley The revolution of new views of the subcellularorganization Simpleand affordable steps to exploitrecent of bacteria. advancesin ffuorescenceimaging. 100 |nvestigating a bacterial killer usingatomic force microscopy 118 HenryBaker: author of the MeganNufiez & EileenSpain first m icroscopy labo ratory Learnabout the secretlife of the predatorykiller, Bdellovibrio. manual Virusesand intracellular RichardBurns This18th century polymath played a big partin popularizing movement earlymicroscopes. TomWileman How do virusesmanaSe to move within and between the cellsof their host? Comment - Openoption for SCM 108 Studyingsingle molecules in journals microbialsystems RonFraser & RobinDunford ChristophBaumann Openaccess demands by grant-awardingbodies Individualmolecules in livingcells can now be observed. arecausing problems for publishers.

{ovr:r inoge Microscopelenses. Tony Craddock / SciencePhoto Library Theviews expressed fditr.rrDrCavinThomas[lcii|*riili{l*"lrdDr5ueAssinder'Professor|ainHagan,ProfessorBertRlma by contributorsare not Ile:;ignfr necessarilythose of the SpencersWood,ReadingRCT1AC Tel.01 18988 1809 Fax01189885656email mtoday6sgm.ac.uk webwww.sgm.ac.uk ,^\rju*rtiti*gDavidLancaster,McMillan-ScottPLC, Society;nor Londonoffce,10SavoyStreetLondonWC2E7HRTe|,o2o7B7B2316Fax02073797118emai|david6mcms|ondon.co.ukf*siiari*it1.*rirtag*lpp'91SGM; canthe CollegeLondon/SPL:125 Mauro Fermariello/SPL;127 Photodisc;129,131 Stockbyte;133 Simon Lewis/SPL;135 Tek lmage,tSPL;139 Digital Vision claimsof advertisers

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Underthe microscope Microbiology Thetheme of this issue To view thesecheck out the scientificsessions there lostfor words is imaging.lt includes www.sgm.ac.uk/pubs/ will be demonstrationsof lne tortoflalboaro oT articlesdescribing micro-today / Yourfeed back techniquesand equipment Microbiologyhas been someof the secretsof on this extrafeature is from a rangeof suppliers. concernedthat manyarticles microbialcells now being welcomeas we will trYto Smallgroups of delegates in the journalwere longer revealedthanks to new providemore animations if will be ableto attendshort than they reallyneeded to technological developments readerslike them. presentationsand alsosee be. Articlesin JCV are already in microscopy,plus a lool< the goodson displayin writlen to strictlength limits, backat a pioneerin the Seemore at York the tradeexhibition. These andthe EditorialBoard of useof microscopeswho Theimaging theme isalso workshopshave been IJSEMhas succeeded in workedwhen the existence continuedin a two-daY scheduledso that everyone recentyears in reducing just of micro-organismswas symposiumat the SCM attendingthe meetingwill the averagelength of beingdiscovered. meetingat York UniversitY be ableto drop in. There arliclesd ramatically th rough Anotherfeature gives in September.lt takesPlace will be somethingof a consistentpolicy of soundadvice on what to on 13 and14 September interestto all, no matler encouragi ng conciseness. lookfor when obtaining andwill includesessions on what theirspecialist field in As an encouragementto equipmentin thisfield. fluorescence and confocal microbiology.See www. brevity,Council has agreed As an enhancementto laserscanning microscoPY, sgm.ac.uk/meetings/ that Microbiologyarticles of (including thisarticle, for the first scanningprobe microscoPY, MTG PAGES/York05.cfm for up to 4,500words time Microbiology TodaYis and electron(cryo) full detailsof the scientific legends,but excluding makingmovies available microscopyand electron programmeand the references)and eighttables or with the web version. crystallography. Between workshops. figureswill be acceptable,but thoseexceeding these limits will be subjectto a chargeof sciencechampion for f.75 (plusthe inevitableVAT SCMwins New where applicable)for every secondsilver Scotlan d additional500 words or parl medal at SCM memberProfessor Anne Gloverof Aberdeen thereofand everyadditional Chelsea Universityhas been appointed Chief Scientific Adviser for displayitem. This policy will Scotlan''d.She has been seconded to the ScottishExecutive come into effectfor articles to provideindependent advice to ministers,take the lead submittedfrom 1 October ,;;;;;,*;;;;;*;; on co-ordinatingscience policy and work closelywith the 2006. Thelevel of chargewill ,isi*,s, on the accepted .t.ffi'::i{}' sciencecommunity. Anne Cloverwas an electedmember of be assessed SILVERMEDAL SCM Councilfrom 1995to 1998. versionof the manuscript.

Address Bool< 2006 ACM 2OO5 A new editionof the AddressBook, containing a listof the cllqlSlA rLowER Sllow 2006 TheACM of the Society fs Gereral Micobiology namesand contactdetails of Societymembers and other Add ro . Society will be heldon Tuesday12 Fy e eshihird lnfomation on dant dheas€s usefulinformation about SCM is beingcompiled and will IN-THELINOLIY RANC4 Septemberat the meeting with the Novemberissue of Microbiology be distributed at YorkUniversity. Agenda Today. TheSCM's display on papers,including reporls let the MembershipOffice have any changesto your microbialplant diseases at Please from Officersand Croup telephone/faxnumbers or emaildetails as soon the RHSChelsea Flower address, Convenersand the accounts possible,but not laterthan 11 August.Send them to Show in May attractedthe as of the Societyf or 2OO5, [email protected] atlentionof thousandsof are in the separatebooklet visitinggardeners. lt also Also,if anyonewishes to havetheir detailsomitted from distributedto all members the Society, with this issueof pleasedthe judges,as You the AddressBook and hasnot alreadynotified canread on p.132. they shoulddo so immediatelY. Microbiology Today.

;.i 90 microbiology today ;i il Staffn ews SCMCouncil May MeetingH igh lights Outgoingand New Council Members ProfessorPeter Andrew, Leicester,Professor Jeff Cole, Birminghamand ProfessorJeff Errington,Newcastle, will sooncomplete theirterms on Council,and their input and contributionswere gratefully acknowledged. There were three nominations so an election wasnot required:the followingwill becomemembers of Councilat the ACM in September: ProfessorMichael R. Barer,University of Leicester,Dr RichardM. Hall,ClaxoSmithl(line, Harlowand Dr CatherineO'Reilly, Waterford Institute of Technology,lreland.

5CM Prizes2007 Councilwas reminded that nominations are invited forthe SCM Prizes2OOT.the Fleming Award,the PeterWildy ?rizefor MicrobiologyEducation, the Colworth PrizeLecture and Congratulations to Staff the FredCriffiths Review Lecture (see Microbiology Today, May 2006 or the SCM website EditorStefan Sidorowicz for details).The closing date is 30 September2005 and the decisionswill be madeby on hismarriage to Helen Councilat their Novembermeeting. Willison the 3 Juneat All SaintsChurch, Sedgley, SCMAnnual Report 2OO5 WestMidlands. Helen is Officerspresented their draft Annual Reportsfor 2005 to Council.These were acceptedand a PAfor a construction a copy of the entireAnnual Reportis enclosedwith this issueof MicrobiologyToday. companyand over 100 peopleattended the Membership wedding.The reception Councilwas encouraged to learnthat a campaignto enrolnew membersfrom different was held at the Beacon areasof microbiologyis underway.The health and diversityof its membershipare among Institutefor the Blind, the core strengthsof the Society. Wolverhampton,and was followedby a disco. Biosciences Federation TheChief Executive of the BiosciencesFederation (BSF), Dr RichardDyer, was welcomed We are very sorry to by Council.He gavea shortpresentation about the aims,business plan and management announcethe deathof of the BSFand answeredouestions from members.The BSF was founded in2002 and John Brimelow,the former wishesto be an authoritativevoice of the biologicalsciences communitywith a big impact ManagingEditor of Journal on policyand decisionmaking atgovernment level. This it intendsto do with the helpof of 1eneral Virology, who its member organizationsbut without duplicatingtheir efforts.SCM hasbeen a member of workedon SCMjournals 85Fsince it wasfounded, and Councilwas pleased to offeran increasedlevel nf c,rnnnrl- from 1985to 2005.John for the nextthree years, provided certain conditions were met. leavesa wife, lsobel,and two children. Ulrich Desselberger,Ceneral Secretary

Newsof Members CeoffreyTalbot Banks(1933-2006), a member since1965. He will be rememberedas a lecturerand fermentation Memberof CouncilPetra Congratulationsto Elected technologistat the Departmentof Biochemistryat lmperial visitingprofessor in the Oystonwho hasbeen made College,London. After a spellat Claxoin BarnardCastle lmmunityand Inflammationat Departmentof Infection, in the early1960s, Ceoff joined ErnstChain's team at the Universityof Leicester. lmperialCollege, where he workedinter alia on interferon TheSociety notes with regretthe followingdeaths. production,publishing several papers in Nature.He retired Dr SidneyR. Elsden,President of the SCM 1969-1972,on from lmperialCollege in the early1990s, settling with his 29 April2006, aged91. An obituarywill appearin the next wife, Mary, in the Valeof the White Horse. issueof MicrobiologyToday. AlexandraSchwenger, a postgraduatestudent at the Dr DavidM. Meredith,5t James'University Leeds Universityof Aberdeen,died tragically in a caraccident (membersince 1980). April (membersince )anuarv 2006).

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ThusRobin's interests New President spaninfectious disease and cancer.Indeed, a recent ProfessorRobin Weiss veterinarystudent in his of Viraloncology at University RobinA. weiss is Professor lab,Claudio Murgia, has in Zoologyat UCLin 1951' CollegeLondon. Robin graduated completeda studYon how Hisfirst research was on the and gainedhis PhD in 1969' a cancercell hasevolved radioactivityon rat populationsin effectsof high natural into a cellularParasite that is hasspent most of his research l(erala,lndia. However, he sexuallytransmitted among includingthe discoveryof careerstudying retroviruses, dogsand hassPread globallY, in a Mendelianmanner endogenousgenomes transmitted d isregard ing transPlantati on with MichaelAbercombie' while stilla researchstudent barriers.Robin also takes laboratoryin Praguein He worked brieflyin JanSvoboda's an interestin the historY as a postdocwith PeterVogt in 1968/9 and spent 2 years of microbiologYand saYs a laboratoryat the lmperial the USA,before establishing thathemuchenjoyedtherecent'historical'issueof lgT2investigatingretroviral on Robert CancerResearch Fund in MicrobiologyToday' He haswritten a new slant 'grandfather 123' 359)' oncosenes.Whi|ehewasDirectorofthelnstituteofCancerl(ochas the of cloning'(Cell,2OO5' he investigatedhuman T-cell Research,London, 1980-1989, and a book reviewon the NobelPrizes in infectious |eukaemiavirusandH|V.Hemadesignificantcontributionsdiseases(Nat Med, 2005, 12, 605)' whichincluded to our earlyunderstanding of HIVand AIDS' neutralizing RobinwaselectedtoEMBOinlgTT,andtotheRoyal the identificationof CD4as the HIV receptor' SocietyinlggT.HewasawardedthelstInternationa| antibodiestoH|V,andthedemonstrationthat.Slim'disease NetherlandsAcademy of Arts he hasconducted BeijerinckPrize of the Royal in Ugandareally was AIDS' More recently' andSciencesin2O0l(SCMmemberDavidBau|combe researchonpigretrovirusesasapotentialinfectionhazardin retires was awardedthe 2nd BeijerinckPrize in 2004). He and on the aetiologyof AIDS-associated xenotransplantation, governingbody of the BBSRC herpesvirus' thisyear as Chair of the malignancies, particu larly l(aposi's sarcoma

NewCrouP Conveners Eu karyotic MicrobiologY ClinicalVirologY DrAlastair Coldman Professor)udY Breuer I obtainedmY first degree Judyis Professorof VirologY in Ceneticsat QMC London at Bartsand the London in 1986.After a sPellas MedicalSchool, Queen a researchassistant, I did Mary CollegeLondon ano my PhD,with Maj Hult6n' head of the Centrefor centredon meiosis,which lnfectiousDisease. Having continuesto be mY main oualifiedin Medicine,she areaof interest.Using the trainedin MicrobiologYand in the DePt new technologYof FISH' up a lectureshiP l(ingsCollege of viraldeterminants ot Virologyat of MolecularBiologY and VZVvirulence. Professor I analysedchromosome and St MarY'sHosPitals. at Sheffield' pairingand segregation BiotechnologY at Breueris headof Clinical Followingresearch The mainemPhasis of work and of the Health in humanmale meiosis. NIMR,she was aPPointed Virology the lab in my lab revolvesaround ProtectionAgencY Varicel la ln 1993ljoined asSenior Lecturer in Clinical on of MichaelLichten at the geneticinfluences Bartsand the ZosterReference Service. Virology at the mode and detailof activelYinvolved NIH where I crossedthe London.Her researcn Sheis also work on DNA double-strandbreak in the establishment speciesbarrier to interestsinclude the We concentratingon the repairduring meiosis. of of clinicalacademic yeast, molecularePidemiologY work on in interactionbetween meiotic alsoundertake Varicellazoster virus (VZV) trainingProgrammes and proteomicand cYtological microbiologYand virologY. chromosomePairing and the Okastrain vaccine, in recombination.I then took aspectsof meiosis Yeast. aswell asthe investigation

microbiologYtodaY *ugg *S 92 Institutefor AnimalHealth, and as Presidentof the British Prize Associationof CancerResearch. Otherwise, he says,he would not havetime to devoteto the SCM. Lectureship 'l Robinwrites: havebeen a memberof the SCM since PeterWildy Prize 1968 and it reallyis a privilegeto tal

microbiologytoday r:ri,tlJ *{1, in departmentalseminar Marinemicrobes star in DVD Crants programmes. Appl ications 'Smallin size,big in importance'is the microbiologists' Overseasschemes will be dealtwith on first single-celled basis mantra.Marine bacteria,archaea, viruses and Thedeadline is 13 come,first served plantsand animalsare not only crucialin makingthe Earlh October20O6 for receipt duringthe academicyear. habitable,but their geneticdiversity provides an immense of applicationsto the use,for novel Education resourcethat we are onlyjust beginningto followingschemes: DevelopmentFund/ productsand processes.Out of the Blue,anew natural International Research PUSAwards DVD by the Ul( Natural Crants Crantsare availableto EnvironmentResearch lnternational membersfor projects Council,will helpto getthat DevelopmentFund intendedto leadto an messageacross to a wider WatanabeBook Fund improvementin the audience.'TVprogrammes teachingof any aspect such as PlanetEarth ignore Studentschemes of microbiology relevant the realaction', explained PostgraduateStudent to educationin the Ul(. PhilWilliamson, the DVD's MeetingCrants Fundingis also available for co-director.'Ad mitted lY, Applicationsfor a grant smallprojects to promote microbesare difficultto film, to attendthe SCM'sYork the publicunderstanding andthe publiconly thinks of meeting (11 -14 September) of microbiology,such them as primitiveorganisms must be receivedby asworkshops, talks, that are agentsof disease. 8 September2005. demonstrations,leafl ets, But they havea lifeof their activitiesat science own, with socialbehaviour, President'sFund festivals. soohisticated biochemistries researchvisits and immenseecological Fourapplications were Applicationswill be importance.Without them, successfulin the lastround; consideredon a firstcome, duringthe neitherpolar bears nor hummingbirds would lastfor long'' the secondround of first servedbasis applicationscloses on calendaryear. Narratedby QuentinCooper, Out of the Blueis produced 13 October 2OO5. for the NERCBlueMicrobe knowledge transfer network RetiredMember (www.bl uem icrobe. com) bY PanacheProd ucti ons' Oxford. Electivegrants ConferenceCrants cellsignalling, Bioprospecting,molecular technologiel Fundi ng for medical/ dental/ Retiredmembers are virusesare coveredand may biofilmsand the versatilityof veterinarystudents to wort< remindedthat they imageryof microbial grant to illustratedby computer-generated on microbiologicalprojects now apply for a minute DVD (playableon PCs processes.Copies of the 35 in their electiveperiods. attendone SCM conference from p.williamson@uea. and PALDVD players)are available Theclosing date for eachyear. The award covers no cost;however, if a reprintis ac.uk.Initial distribution is at applications is 27 October en-suiteaccom modation be made. needed,a no-profitcharge may 2005. and the Societydinner, uP to a maximumof f250. Otherschemes Conferences2A06 Applicationsfor grantsto Careers SeminarSpeakers Fund attendthe SCM meeting 4 November Universityof Manchester TheFund supports talks at York Universityare now 18 November Universityof Edinburgh on microbiologicaltoPics invited. 2 December UniversitYof Reading

Aimed at life scienceunder- and postgraduatestudents, eachconference includes widely varying talks on career choicesand furthertraining, plus a smallexhibition by a rangeof organizations.A CV review serviceis also available by priorarrangement. SCM is involvedin organizingthe eventsand will havea standin the exhibition'Cost f12,to includerefreshments and lunch.Details and a bookingform are availableat www.bsf.ac.uk/careers/careersconf.htm

today*ug CI6 94 microbiology FI' l"

TheU l( ClinicalVirology Network (CVN) Elected TheCVN hasaround 30 membervirology laboratories. The CVN website(www.clinical- Fellows virology.org)contains a wealthof informationon virologicaltopics anda messageboard. of the Royal Additionally,the networkoperates a web-basedcontinuing education tool called5ClQAS. Society SCIQAS Thefollowing SCIQASpresents the userwith a web-basedinterface to virologyclinical scenarios. Six microbiologistshave been distributionsperyearwill be provided;each will containtwo scenarios.Each is a real electedas new Fellowsof clinicalcase submitted for inclusionby one of the CVN'sspecialist virology laboratories. the RoyalSociety. SCIQAS'primary aim isto provideparticipants with a method convenient of augmenting NicholasJ. White, OBE, virology their knowledgeof clinical and increasingtheir ability to accuratelyinterpret Professorof Trooical clinicalinformation. Medicine,University of On login,users are asked to choosea Oxfordfor outstanding scenariofrom the currentdistribution (right). contributionstowards Therelevant scenario is displayed,including an understandingof the clinicalinformation on the patientin question, pathoge n es is, treatme nt the samplesubmitted and assaysperformed and control of tropical upon it. An examplescenario is displayed d iseases. below. Joseph5.M. Peiris, Professorof Microbiology

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t*uili$rilnth-$i**s of Microbiologyat the Universityof lllinois Theuser then inputstheir clinical hasbeen appointed a interpretationof thesedata; simultaneously New ForeignMember jsuvvaxttcfdE they,maystate that additionalsamples ought j*.*rotdr*t*'n"t- of the RoyalSociety. He to be takenor further performed Iou,*r*orr"r,o assays on is distinguishedfor his irelrhdk

lmmunofluorescencemethods, together with CFP anddigital imaging, are providing a revolutionarynew organizationof bacteria,as Jeff V Technicianusing a viewof the subcellular fluorescence microscoPe. CCStudio ,/ SciencePhoto Library Errimstsn d escri bes.

acterial cells are remark- able in many waYs. TheY carry oul a huge and diverse range of biochem- ical reactions and are capable of growing in an amazrrrg range of environmenlai con- d.itions.One secretof their successlies in the adaptability that comes from their rapid growth and division. This comes, in turn, from their small size and. simple architecture, compared with cells of higher organisms' When the electron microscope was developed in the mid-20th cenlury, and applied to bacteria,it confirmed the notion that bacterial cells are simple. There was no sign of the complex, membrane-bound organelles that charactertze eukaryotes' However, there is a sense in which these experiments did a disservice to bacteria, because they emphasized aII - of the things theY were missing cytoskeleton, membrane-bound organ- elles, particularly a nucleus. Meanwhile, some bacteria, such as Eschenchiacoli and B acillussub tilis,b ec ame great exp eri- mental work-horses because of their rapid and easy growth and excellent genetics.Many scientistsbegan to view 'bags them essentiallyas of enz)'rynes'- wond.erful for biochemical studies and as tools for molecular biology, but of little interest from the perspective of celluiar organization. This view changed dramaticallY in the mid-1990s when the renewed appli- cation of immunofluorescencemethods to bacteria, together with the advent

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of green fluorescent protein (GFP) and digital imaging, it to be detected by any of a number of readily available provided a revolutionary new view of the subcellular organi- monoclonal antibodies.The main disadvantageof this method zalron of bacteria. These developments founded what was arises from the need to get large antibody molecules into 'bacterial effectiveiy a new field of cell biology' and provided the cell. To achieve this, the cells need to be permeabrlized. a powerful suite of methods and approaches with ivhlch to Since this would normally result in destruction of the cells, probe fundamental aspectsof the workings of bacterial cells. they fi.rst need to be gently fixed. But this is tricky because too much fixation makes the cells impenetrabie. Therefore, Fowerfuinew tech nologies the whole process is technically demanding. Inevitably, the Fluorescencemicroscopy had been applied sporadically to problems of accessof the fluorescent label and degradation bacteria in various ways for 30 years or more. However, in of cellular structure mean that artefacts and even compiete the early 1990s it entered the mainstreamwith the successful failure of detection are not uncommon. Finally, since the application of immunofluorescence methods to intracellular cells are obviously killed by the process, artefacts can arise proteins in bacteria, followed soon after by the harnessingof from cell death and there is no question of being able to GFP At around the same time, the development of affordable follow movement or changes ln localization over time. CCD cameras with high sensitivity and spatial resolution, GFP is an intrinsically fluorescent protein derived from a togetherwith digital imageprocessing packages, made it much jellyfish, Aequoriavictona.The big advantageof GFP labelling easierto capture the faint fluorescencefrom bacterial cells. is that it can be done in native, unperturbed cells. Moreover, Immunofluorescence methods essentially involve the use the cells can be imaged repeatedly, offering the possibility of a fluorescentiy labelled antibody to identify the location of of collecting optical sec[ions through cells, or obtaining a the target protein. A major advanrage of the method is that time lapse series. Although GFP has had a revoiutionary it can be done with completely wild-type cells, provided a effect on research across blology (of which more below) specific antibody is available.In principle, the location of the there are some caveatsand limitations to its use. The main fluorescentantibody repofts the position of the native protein. disadvantage arises from the fact that the cells need to be Aiternatively, the cells can be genetically engineered to add genetically engineered in some way, to produce a fusion a shon (- 10 aa) epitope tag to the target protein, allowing of GFP and the target protein. Many, perhaps most, fusion

microbiologytoday aug i){: 97 l Bacillussubtilis cells carrying a CFPfusion to the SpoOJchromosome segregation protein. Thisprotein sticks to regionsthat flank the originof replicationof the circularchromosome andforms spots or'foci'(green) that markthe originregion. The spots duplicate soon after chromosomereplication begins, as soon as there are two copiesof the originregion. These then moverapidly apart towards opposite poles of the cell.The chromosome (blue), occupies mostof the celland the foci lie at eachend of the DNA mass.The cell outline is stainedrec with a membranedye. Two pairs of cellsare shown. The cells are about 2 pm long. Dr HeathMurray proteins are not completely wild-type in behaviour, because which are involved in mapping the size and shape of rhe cell adding the 27,000 Da GFP moiety can mess up the target for the purposes of DNA equipartition or accurate selection protein's activity in any one of a number of different ways. It of the mid-celi site for cell division. is therefore important to check that the fusion is functional, Overall, the results show that bacteria have an astonishing by testing for its ability to sustain the function of the normal and completely unexpected level of complexity The patrerns gene, where possible. If the protein is not fully functional, being distinguished today probably represenr rhe rip of the any pattern of locahzation should be treated with caution. In iceberg, becauseresolution is currently confined by the physr- our experience the two most common problems that arise cal limitations of fluorescence in the visible light range. A 'inclusion are: fi.rst, aggregation of the GFP fusion to form gre ter level of sophistication probably awaits discovery when bodies', often in the form of a single bright spot located near emerging technologies that can overcome the current limits the pole of the cell; second, loss of a specific pattern because of resolution become availableto bacterial cell biologists. of saturation, due to the fusion prolein being overexpressed relative to the native protein. JeffErrington FRS A good way to mitigate being misled by this kind of Dir"ectorof the Instituteof Celland Molecular problem is Lo construct GFP fusions to both the N and C Biosciences,Faculty of MedicalSciences, University of [erminus of the target protein. Sometimes only one of them Newcastle,Newcastle upon Tyne NE2 4HH, is functional, and it is not uncommon for the two fusions Ul< ([email protected]). to give different resultsl In our experience with hundreds of Jeffis currently setting up a fusions, the N-terminal fusions have a slightly better success world'sfirst'Centre for BacterialCell Biology'to reflect rate, which is unforlunale becausethey are somewhat more the importanceof thisnew field for microbiology. difficult to construct (the gfp gene has to be placed between the coding and upstream regulatory sequenceof the gene). Furtherreading Although GFP is often used as a reporter of subcellular Ausmees,N., Kuhn,J.R.&Jacobs-Wagner, C. (2003).The localization, it can also be used as a repofter of the timing bacterialcytoskeleton: an intermediatefi lament-like functio n. Cell or celi-specificity of gene expression. Fusing gfp to the rr5,705-713. regulatory sequences of one's favourite lene provides a way Errington,J. (2003). Dynamicproteins and a cytoskeleronin of identify'rng where and when the gene gets turned on. This bacteria.Nat Cell Biol 5, 175-I7 8. can be very informative in multicellular situations, such as Jones,LJ., Carballido-Lopez,R. & Errington,J.(200I). Control in biofilms, or to study genes turned kind on under any of of cell shapein bacteria:helical, actin-like filaments inBacillus stochasticcontrol. subnlis.CeIl I04, 9 13-922. (1998). A myriadof patterns Lemon,K.P & Grossman,A.D. Localizationof bacterial DNA poll.rnerase:evidence for a factorymodel of replication. GFP fusions have now been made to many thousands of Science 282,1 5 I 6-1 5 19. bacterial proteins. An ever-increasing multitude of different Lewis,PJ., Thaker, S.D. & Errington,J.(2000). localization patterns has been described. Some of the resulb Compartmentalizationof transcriptionand translationin Bacillus have been of a unifying nature. For example, it is now clear subtilis.EMBO J 19, 710-718. that [ranscription occurs in the core of the cell and translation (199I). in the periphery, just as in eukaryotes, even though there is Lutkenhaus,J. FtsZring stmctureassociated wrth divisiontnEscheichia no nuclear membrane demarcating these zones in bacteria. coli.Nature 354, 16I-164. 'replication DNA replication occurs in specialized factories', Moller-Jensen,J.6r Lowe,J.(2005). lncreasing complexity of just as in eukaryotes. Localization experiments have also been the bacterialcytoskeleton . Cun OpinCelI BioI 17 , 7 5-Bl . crucial in demonstrating that bacteria have all three elements Raskin,D.M. 6r de Boer,PA. (1999).Rapid pole-to-pole of the eukaryotic cytoskeleton, tubulin (FtsZ), actin (MreB) oscillationof a proteinrequired for directingdivrsion to the and intermediate filament proteins (crescentin), with criti- middleof Eschenchiacoli. Proc N atl AcadSci U 5 A 96,497 1497 6. cal roles in the cell cycle and cell morphogenesis. Among Wang,X., Possoz,C. & Sherratt,DJ. (2005).Dancing around the surprises have been the discovery of proteins that rapidly the divisome:aq.'rnmetric chromosome segregation in Escheichia oscillate,from pole to pole, or round and round the cell, and coli.Genes Dev L9,2367-2377 .

9B microbiology today;lug 06 bacteria and eukaryoles. Becauseof this broad yet specific spectrum of target organisms, the role of bdellovibrios in the environment and potential applications in industry, agn- culture and biodefence are of particular interest. In the past couple of years, we have explored the predator's behaviour at interfaces and surfaces with an eye towards examining its role in biofilms and other complex microbiological communities. Atomic Force Microscopy (AFM) has proved especially useful in exploring the interaction of bdellovibrios with prey in these environments.

UsingAFM With AFM, a small, pointed tip is scanned acrossthe surface of a sample.Small deflectionsand alterationsin the behaviour of this tip are then used to generate an image of an object's he lifestyle of the bacterial predator Bdellovibno shape and contours. In general, AFM can resolve features bactenovorusbears a striking resemblance to [he on the nanometre to micron scale.With soft microbiological creatures in the Alien mowes. Propelled by its long samples such as our predator and prey cells, we have used polar flagellum, this nanoscale killer swims about AFM to resolve small structures on individual cells, such as randomly until it encounters aprey bacterium. When scars,flagella and pili, as well as to study groups of bacteria it finds and identifies a prey cell, the bdellovibrio at once (Fig. l). predator burrows through the outer membrane and takes AFM provides high-resolution information about the sur- up residence in the prey periplasm, sealing off the outer faceof a sample, so we might not expect it to reveal anything membrane behind it. The bdellovibrio excre[esa variety of about the interior of a cell. However, it turns out that we enz4es to digest the prey cell, growing larger and larger can in fact learn a lot about the interior of bacterial cells as as it breaks down and assimilates the prey cytoplasm. At well. For example, we can clearly distinguish the growing this stage in the life cycle the growing predator protected by bdeilovibrio predator inside of the periplasm of an invaded the carcassof its prey is called a bdelloplast. When the prey prey ceil. The shrunken ball of cytoplasm also can be cytoplasm is totally devoured, the predator cell divides into discerned (Fig. 2). progeny cells that lyse the outer membrane and swim away Thus AFM provides a useful complemenl to traditional to find some new cell to consume. forms of microscopy, given that its resolution is better than B. bactenovorusconsumes a wide variety of Gram-negative light microscopy, but that samples require minimal or no prey, but is harmless [o other bdellovibrios, Gram-positive preparation compared to electron microscopy It is important lr-rvestlgotr-lI o booterlol er rJslrro otor-Ttle foroe lnIorosoopy

100 microbiologytoday aug 06 to note, however, that the cells of interest must sit on a soiid surface to be imaged. Given the importance of surfaces and interfaces to bacterial growth, this requirement has provided little difficulty and also new areas for exploration.

Studyingcommunities We initially grew simple bacterial communities on sterile polycarbonate filters placed on top of agar medium plates, an environment perhaps best described as a hydrated air- solid interface. Both prey and non-prey organisms, including severalstrains of Eschenchiacoli, Aquaspinllum serpens, Micro- coccusluteus, Ensifer adhaerensand mutant host-independent Bdellovibno, grow robustly, and divide and spread outward across the surface of the fi1ter.The cells can be easily imaged by AFM right on the filter without modification. Interestingly, when bdellovibrio predators are mixed with a Gram-negative prey population, they thrive in these surface communities. Within the first few hours after deposition onto the filter, only prey cells are seen, but soon the first bdelloplasts appear. The predators and bdelloplasts multiply and within a few days the bdelto- vibrios completeiy dominate the surface, forming a contin- uous lawn of large, well-fed predators. No prey cells are lefi

A Fig.1. AFM image of E.coli Zl<1056on a glass surface. With this technique,bacteria can be imaged without staining or dryingunder vacuum,and structures such as individual ffagella and pili can easily beresolved. Modifed from Nifiez et al.,(2005) Methods Enzymol 397,258, with permission from Elsevier.

{ Fig.2. Despitethe fact that AFM is by nature a surfacetechnlque, thecurved shape of theBdellovibrio predator is clearly visible growinginside the ruined, rounded carcass ofthe prey cell. Modifed from Nifiezet al.,(2005) Colloids Surf B 42, 267,with permission fromElsevier.

Atomicforce microscopy is a powerfultechnique for studying biologicalsurflaces and molecular structure.In thlsarlicle Megan Nfifiezand Eileen Spain discuss the applicationof AFMto investigate the interaclionbetween predator bdellovibriosand their prey. microbiologytoday aug 06 A Fig 3. Bdellovibriopredators devour E. coli alive except for a few refugees found interfacial environments, in par[icular (a) the preycells preyon a surface. Initially, at the outside edges of the bacterial within biofilms. We prepared simple divideto coverthe filtersurface densely. (Fig. 'biofilm- (b) After 1-2 days,Bdellovibrio predators community 3). model biofilms by growing a and bdelloplastsbegin to appear.The The astounding success of bdello- forming' strain of E. coli, ZKl056, on bdelloplasts,such as the threeindicated vibrios in these surface communities glass coverslips half-submerged in by arrows,can be distinguishedfrom the raises several interesting queslions. In growth medium. These E. coli prey unmolestedprey cells by a roundedshape, a smoothsurface texture, a shrunken pafticular, we wonder about the nature cells formed a dense, matted film on cytoplasmand a distendedperiplasm of the air-filter interface. How thick the glassat the airJiquid interface. AFM inhabitedby the oblong,coiled predator is the layer of liquid above the fiiter? revealed that these cells adhere to [he ceils.(c) Within 3-5 days,the surfaceof hunt on a glass in clumps, utilizing multiple pili the filteris entirely covered by the oblong, How do the bdellovibrios vibrioidpredators. Fig. 3b is modifedfrom surface?Do they swlm or just wiggle? and flagellaas well asal^yer of excreted Nifiez et al. (2003)Biophys J 84, 3383, It is believed that the force generated sticky extracellular material. As on the permission the BiophysicalSociety. with from by the movement of the flagella and frlters we found that the predators the collision with the prey cell is import- hunted quite successfully within this I Fig.4.AFM images of bdellovibrios t attackingmodel biofilms. Unlike staining ant for penelration of the prey cel1, surface community, devouring most or methods,AFM allows us to distinguish but how much force can be generated all of the cells and leaving little behind normalprey cells from predators, on a flat surface within a tightly but debris, evenwhen the prey cellshad bdelloplastsand celldebris. (a) The biofilm- packed communily? Bdellovibrios 24hours to establish[hemselves before formlngE. coli strainZl<1056 attaches to a glasssurface submerged in liquidand forms clearly dominate these communities, the predator was added. Within these communitiesthere. (b) In this interfacial consuming all of the prey cells and simpie biofilms, protective features, predator environmentthe bdellovibrios growing Lo extraordinary lengths, un- such as excreted extracellular ma[erials attackand devourprey cells. Modif ed from in liquid and compact clumps of cells, do not Nifiez et al., (2005) ColloidsSurf B 42, Iike in the wild or even 266-268, with permissionfrom Elsevier. medium in the laboratory Is their suc- seem to protect the prey ce1ls from cess due to the benefits of a 2-dimen- the bdellovibrio predators. Neither do sional (as opposed to a 3-dimensional) slow-growrng prey escapethe predator, search? Do they receive additional as they might escapeantibiotics. Only nutrients by'grazing' on cell debris? in more concentrated, sugary growth medium can the prey cells reach equili- Bdellovibriosin biofi lms brium with the predators(Fig. a). Given the success of bdellovibrios in hunting and devouring prey com- Otheruses of AFM munities on the hydrated air-solid Thus far we have primarily exploited the interface of a filter over a culture plate, imaging functions of the atomic force we began to wonder how successfully microscope to investigate Bdellovibno the oredator would hunt within other predation. Notably, other investigators

m icrobiology today i:"r-{i;{-}{i; AFM provrdesa usefulcomplement to traditionalforms of microsco py, given that itsreso Iutio n is better than Iight microsco py, but thatsam ples requireminrmal or no preparation compared to electronm icroscopy.

have used AFM in different and eleganr ways to expiore Furtherreading their favourite microbes. Some have probed structurls of Dufr€ne,Y. (2002).Atomic Force Microscopy, a powerfultool subcellular and molecular assemblieson cell surfaces and in microbioiogy.J Bactenol I84,5205_5213. freeze-fractured membranes, including porins, S-layers Kindt,J.H.,Sitko,J.C., pietrasanta, L.I., Oroudjey E., and the purple membranes of Halobactenum.Orhers have Becker,N., Viani,M.B. 6r Hansma,H.G. (2002).Methods elucidatedthe effectsof environmental factorsand antibiotics for BiologicalProbe Microscopy in aqueousfluids. rn AtomicForce on cellularinregdry by repeatedlypoking microbial cells wirh Microscopyin CellBiolog,vol. 68, p.2I3. Editedby L. Wilson, the AFM tip to measurethe cells' physical properties. other P Matsudaira,B.P Jena 6JJ.K.H. Hoerber. London: have quantitatively Academic $oups measured forces of adhesion Press. between specific cells, surfaces and molecules. Another Lower,S.K., Hochella, M.E,Jr & Beveridge, (2001). interestingapproach to biologicai exploration with AFM is TJ. Bacterialrecognition of mineralsurfaces: nanoscale to measurethe real-time dynamics of celluiar processessuch interactions 'breathing, betweenShewanella and cr,FeOOH.Science 2g2,1360_1363. as division, motility, contraction or occurring in solutionor atahydrated interface.This kind of measurerrrent Martin, M.O. (2002).predatory prokaryotes: an emerglng research has been used successfullywith eukaryotic cells,"but less oppoftuniqzJ MolMicrobiolBiotechnol 4, 5_16. frequently with prokaryores. Nriflez,M.E., Martin, M.O., Chan, pH. & Spain,E.M. Finally, there are many variations of scanning probe (2005).Predarion, death, and survivar in a biofilm:investigating microscopy,including AFM, and also electrochemical force Bdellovibioby Atomic ForceMicroscopy ColloidsSurf B 42, microscopy and magnetic force microscopy that may have loJ-z I I. someapplications to microbiological systems.we expect to Pelling, A.E., Sehati,S., Gralla, E.8., Valentine,J.S.& employ some of this large array of techniques to learn more Gimzewski, J.K. (2004). Local nanomechanicalmotion of about the ways in which Bdellovibnofinds and consumes its the celi wall of Saccharomycescerevisiae. Science 305. prey LT47_II5O.

MeganE. Nrifrez ClareBoothe Luce Assistant professor of Chemistry, MountHolyoke College, South Hadley, MAO1O75, USA(t +1 413538 2449;f +1 413538 2327.. e [email protected])

EileenM. Spain AssociateProfessor of Chemistry,Occidental College, LosAngeles, CA90041, USA (t +1 323 259 2940: f +1 323341 4912; e emspainpoxy.edu)

microbiologytoday ;lr-t;; *S How do viruses,which haveno independent meansof locomotiofl, manageto move withinand between the cellsof theirhost? TomWileman uses fluorescence microscopy to find out. Vlruses or-r d lntroeellulor r-nover'r-ler-t t

he discovery of a dead Whooper exploit intracellular traffi.cking path- their way across these relatively long swan infected with the patho- ways ^t the beginning of what can distances. The cytoplasm is crowded genic H5Nl slrain of avian influ- become a global migration. with organelles and cytoskeleton, and enza on the shores of Scotland Vrruses ate obligate intracellular hypothetical calculations suggest that in April 2006 illustrated how parasiles and need to enter the cyto- viruses would move very slow1y if viruses can move globally in a plasm to gain nucieotides and amino they had to rely on passive diffusion. very short period of time. As pandemics acids necessary for genome replica- It would take at least 200 years, for develop, viruses are distribuled over tion and capsid synthesis. Efficient example, for herpesvirus capsids to long distancesby hosts such as migra- replicarion olten requires transport travel just 1 cm along an axon from tory birds, insect vectors and unwitting of the virus through the cytoplasm to sensorynerve endings to their favoured airline passengers,or passivelyby rivers specific intracellular compartmenrc. site of replication in the nucleus of and other waLer systems. A11viruses, Since viruses have no independent the cell body Interestingly, recent dis- however, start life inside cells, and means of locomotion, it has been a coveries made using microscopes to recent work is unravelling how they challenge to work out how they find observe the movement of fluorescent

104 { lmmunoffourescencemicroscopy revealsthe nucleus (blue) microtubules (green),and actin filaments and stressfibres (red). Bill Rennie, br6tbrp.co.uk

viruses in living cells. The fluorescent structural proteins are either expressed directly from viral genomes by generating recombinant viruses, or expressedin cells pdor to infection, and incorporated into virusesduring assemblyThe trafficking of viral genomescan also be studied by fusing GFP to appro- priate DNA- and RNA-binding proteins. Incorporation of GFP into herpesvrruseshas been particularly successful,and viruses with red capsids and green tegument proteins have been imaged as they travel within neurones in culture. It rs not always easy to produce GFP-labelledviruses, and it has proved particularly difficuit Loengineer naturally fluorescenr icosahedralviruses where capsid geometry is constrained. In some casesviruses are labelled using fluorescent chemicals before they are added to cells, and this has been used ro follow the cell entry of adenovirus and influenza virus.

Virusesuse microtubules and molecularmotor virusesin living cells show that they use the cytoskeleton to proteinsto movewithin cells movewithin, and between,cells. Many DNA viruses replicate in the nucleus, while others, such as poxviruses and many positive-strand RNA viruses, Observingfluorescent viruses in livingcells replicate in the cytoplasm in virus factories, or rearranged 'virioplasm'. Greenfluorescent protein (GFP) technology was first used cellular membranes called These stmctures in the mid-1990s to track and quantify the movement of are invariably close to the microtubule-organizing centre cellularproteins. GFP from the jel1yfishAequoreayictonawas (MTOC) which anchors microtubules close to rhe nucieus. fusedto proteins of interest, and the na[ural fluorescenceof Early studies showed that the replication of many viruses GFPallowed the proteins to be tracked in real time in living was blocked if microtubules were disrupted, suggestingthat cells.Systematic mutagenesis of GFP has produced brighter transport down microtubules towards the MTOC was import- and more stable GFPs, and fluorescent proteins of different ant for them to reach sites of replication. Recent iive-cell colours,allowing severalproteins to be tracked at the same imaging experiments have confirmed that this is true and time. The fusion of GFP and its variants to viral structural provided further information about dynamics of virus proteins has made it possible to follow the movement of movement, and the proteins involved. Live-cell analyses

microbiologytoday ;rr"rq {J{:i ) This page.Summary of viral movement through a cell. Viral core particlesleave endosomes(1), bind to microtubulesand dynein motors (2), and are transported to the MTOC (3). Virus replicationoccurs in the nucleus(4) or in cytoplasmicvirus factories(5). Newly assembledviruses bind l

)' Opposite page.African Swine Fever vi r u s associateswith m icrotu bu les. Virus particles(red) moving through the cytoplasmalign along microtubules (green) Reproduced with permission from Jouvenet et al. (2005) J Virol 78, 7990 (American Society fo r Mi cro b i o Iogy)

are now available for adenovin)s, Adeno-associatedvirus 2, Virusescan use actin to leavecells infiuenza virus, HIY, Vacciniavirus,Afncan swineJever virus Viruses delivered to the cell peripheryby kinesin motors are and herpesviruses. depositedinto the ce1lcortexjust below the plasmamembrane Viruses align on microtubules and move at speedsvarying which conuins high concentrations of actin. Poly'rnerization between I and 4 pm s-t. Many viruses show complex of cortical actin allows cells to produce extensions called bidirectional movement, making it difficult to understand lamellipodia and filopodia which are used for cell movement. how they get from one end of the microtubule to the olher. Some viruses (and severalbacteria) use the power generated Careful analysishas shown that run lengths towards the centre by the polymerization of this rich source of actin to generate of the cell predominale, resulting in preferenlial movement extensionsand to move into neighbouring cells.Vacciniovirus, towards the MTOC. Once virus replication and assembiyare for example, fuses with the plasma membrane but remains completed, it is important for many viruses to embark on the attached to the outside of the cell where it signals the reversejourney to the cell surface. Again live-cell imaging polymertzation of a comet-shaped actin tail which propels has shown that viruses move aiong microtubules, this time the virus away from the cell surface.Afncan swineJever virt+s, towards the plasma membrane, and in some casesviruses a virus related to poxviruses, is also delivered to the actin can switch from one microtubule to another. cortex by kinesin motors where it stimulates actin filament Cells use two kinds of ATP-fuelled molor protein lo move formation. The filaments are bundled beneath the virus and organellesand other cargoesalong microlubules. The dlnein carry the virus away from the plasma membrane at the tip motors move cargoes inwards towards the MTOC while of filopodia. Understanding how viruses induce localized the kinesin motors move in the opposite direction to- polymerization of actin at the cell surface is not only of wards the plasma membrane. Analysis of the sequences interest to virologists. The strong similarity between virus- available for virus genomes made it unlikely that viruses induced projections and the lamellipodia and filopodia used encode their own motor proteins, and since they move at for cell motility allows the study of virus motiiity to provide the same speed as cellular cargoes, it was suspecled that valuable insight into how cells migrate, for example during viruses hr.lackedcellular motors lo move in cells. It is now recruitment of lymphocytes to sitesof infection, meustasis of known that viruses use d1'nein motors to reach sites of repli- cancer cells and outgrowth of neurones. cation in the cell interior, and that after replication and assemblynew viruses switch to kinesin motors to reach the Thefuture of virusmotility cell surface.How viruses recruit the motors, switch them on Advances in mlcroscope technology, such as increased and off and exchange one for another are key areasof current sensitivity and improved imaging rates, coupled with more research. stablefluorescent probes, will allow singlevirus particlesto be r Thestudy OI VtrUSft|t )ttlity will provide valuableinsightinto how cellsmigrate, for example du ring metastas ts of cance r cells.

105 micrO-',biology today ,,lr"r;li.iiii tq

trackedin greaterdetail. It is anticipated by preventing actin mobilization at the Referenceswith videos that dual and triple labelling studies cell periphery. This raisesthe hope that of virusmovement in living coupledwith fluorescenceenergy trans- in the future we will be able to stop cells fer (FRET) and fluorescence recovery viruses in their tracks in cells, before Lakadamyali, M. & others (2003). after photobleaching (FRAP) micro- they start their global migrations. Visualizing infection of individual scopy will be used to follow the ex- influenza viruses. Proc NatI Acad Sci changeof specificmotor proteins as the TomWileman usA 100,9280-9285 (or virusesbind to changedirection on) Schoolof Medicine,University Luxton, G.\M 6r others(2005). microtubules, or switch to actin-based of EastAnglia, NR4 7TJ, Ul< Targetingol herpesvirus capsid motiliqr It will also be imporranr ro transportin axonsis coupledto (et.wi leman @ uea.ac. uk) determine how cellular signalling pro- associationwith specificsets of teins regulate virus motility, and how tegumenlproteins. Proc Natl Acad Sci Further vrrusesswitch them on and off. Work reading u s A\02,5832-5837. Coyne, C.B. 6r Bergelson,J.M.(2006). overthe last year has shown that move- Luxton, G.W 6s others(2006). Virus induced Abl and F1n kinase signals ment of herpesviruses along micro- The pseudorabiesvirus - 'P-I/2 tegument permit coxsackievirusentry through tubules can be reconstituted in vitro. protein is requrredfor intracellular epithelial tight junctions. Cell 124, Suchsystems will allow virus movement capsidtransport. J Virol 80,20I-209. I t9-I3I. to be analysedin biochemical and bio- McDonald,D. & others(2002). physical terms. This will tell us how Dohner, K., Nagel, C.-H. & Visualizationof the intracellular (2005). much force is required to move a virus Sodeik, B. Viral stop and go behar,rourof HIV in living celis. along microtubules: taking a ride with along microtubules, and how many J CeIlBioI 159, 44I-452. dy'nein and kinesins. TrendsMicrobiol motorsare needed. Munter,S., Way, M. 6c Frischknecht, 13,320-327. lt is impoftant to remember that E (2006).Signalling during pathogen many viruses pose a serious threat to Greber, U.E 6s Way, M. (2006). A super infection.Sci STKE 2006 (335) re5. mankind and it is hoped that studies on highway to virus infection. Cell I24, doi: I 0.I 126/stke.3352006re5 74r-754. virus motility can lead to new antiviral Smith,G.A., Gross, S.P 6c Enquist, drugs. One such drug, the c-Abl tyro- Reeves,P M. & others (2005). L.\M (200I). Herpesr,rrusesuse sine kinase inhibitor Gleevac, was Disabling poxvirus pathogenesis by bidirectionalfast axonal transport to shown recently lo prevent the spread inhibition of Abl-family tyrosine kinases. spreadin sensoryneurones. Proc NatI of. Vacciniavirusand coxsackievirus B NatMed 7L,73I-739. AcadSciUSA 98,3466-3470.

microbiologytoday au,4 {-r# ?r Coloured transmissionelectron micrograph of transcription complexesfrom Escherichiacoli on DNA (blue horizontal line). During DNA transcription,complementary mRNA strands(blue vertical lines) are synthesized. Dr Elena Kiseleva/ SciencePhoto Lib rary

Recentadvances in imaging technologies have enabled researchersto pinpoint individual ffi *wd ,Y3" moleculesin livingcells, as <*,; '{W'nf V,uii!l-',:t'{i}Y't,ffiii::u,r:l7i'ffii,ft.'ffiTT deSCfi beS .

n 1683 Antony van Leeuwenhoek pubiished the first which we live. Imagine standing in a crowded room where microscopic description of bacteria, which he isolated people are standing elbow-to-elbow and trying [o move from the plaque between his own teeth. He acquired acrossthe room while the floor beneath you is jiggled violent- the images using a simple magnifi.cation device ly. Your attempts to move in a directed way wiil be scrambled with a hand-ground lens. This important milestone by frequent collisions with your neighbours. Thrs resembles 'thermal highlights a common occurrence in the microbiological the effectsof noise' (Brownian motion) and molecular sciences- significant advancesin our understanding of the collisions on single molecules, which together drive the pro- microscopic world are often linked closely to technological cessof diffusion in a liquid by pushing molecules in random developments. in the last three decades,advances in laser directions. This is a good approximation of the environment and light detection technologieshave allowed us to quantify where enzyme molecules must function and the physical the small dispiacements,and associatedforces, generated by obstructions to movement that they must overcome. a single onzyme molecule. This article aims to make clear the At first glance,the'noisy'environment inslde a cell appears need for single-molecule studies on microbial systems,and an inhospitable place for any molecular process.Furthermore, to highlight some recent milestones in this area. thermal noise increaseswith temperature, thus it is amazing that certain microbes can flourish under extremely hot llsrr ,,-;::'r;J.]',tlt;ir;' *ii{l ,:}{;1 :,i;';:;,.i::;':rr*,lr:.f"ttlf: conditions. In fact, it is thought that an onzqe can use the 'nanoscopic' The nature of the environment where an enzqe energy releasedfrom a chemical reaction to bias the thermal must function is very different Lo the macroscopic world in forcesacting on it and drive mechanical motion.

108 microbiolo gy today ,i.,"jli'i.i{i If this occurs, RNAP should be able to generateforce and do'work'(work = force x distance moved). I *-4 y To determine if RNAP could do work, Studylrr \."- researchers tethered an appropriate e DNA template between an E. coli RNAP immobilized on a surface, andamicron- sized bead held in optical tweezers. Optical tweezers use photon pressure 'trap' ng to a micron-sized particle at a spot in solution where a laser beam is focused. When transcription was restarted, the RNAP pulled on the DNA ECLJCS tether and displaced the bead held in the rno optical trap (Fig. 1). These displace- ments were converted into linear velo- cities of - I0 bp s-1,or -3.4 nm s-t. By increasing the force against which ln rr-tleroblel the RNAP must act, the researchers were able to stall transcription. This force, -25 picoNewtons (pN), is equi- valent to the weight of -25 red blood cells. These were the first experi- syster-ns ments to show that RNAP is able to do work and thus can be considered a 'molecular Here, two k y bacterial enzpe work on gene expression at the single- motor'. complexesare used to showhowsingle- cell levei is used as an example of this A simple model for RNAP movement molecule-level investigations have approach. along the DNA template might pre- 'nano- advanced our understanding of dict that this molecule should move scbpic' motion: Eschenchia coli RNA Mechanicsof gene in discrete -0.34 nm steps, which are polymerase and the bacterial flagellar transcription equivalent to the distance between two motor. These examples are typical of Tianscription of a gene into RNA is a base pairs. Recent technological im- 'molecular two classesof motors', which vital biological process as it is the fi.rst provements have allowed this discrete are charactenzed by their ability to committed step in gene expression. The stepping to be observed at the single- couple a favourable chemical reaction enzpe responsible for transcription molecule level. The steps observed are 'linear' to directed and'rctary' motion, in bacteria, RNA polymerase (RNAP), consistent with E. coli RNAP moving respectively Using state-of-the-art phyr- must move aiong the DNA template. forward I bp for every ribonucleotide ical techniques, the motion of these Due to its relatively large physical triphosphate incorporated into the tiny motors can be observed in real size, an RNAP molecule may pull the growing RNA chain. This resuhimplies time and provide new insight into their DNA through its active site during RNAP couples polymenzation directly -mechanisms. Advances in fluorescent transcription, rather than moving to forward translocation along the DNA imagrng have allowed single molecules along the DNA and pushing through template, which is consistent with a to be observed in living cells. Recent the crowded cytoplasmic environment. Brownian ratchet type model.

obiologytoday aug 06 109 {r, iii ij *:{.t:"::::,I lr: n

'2...j i'r .t,i::.ri; "-i .:

'.i.ri f:ij

"-,; 1 r:1i't'l Magnifiedbead image is observedfrom above fhe DNA duplex is composed of two complementary strands running in opposite directions, which coil around a helix. To if Beadin opticaltrap is pulled common axis to form the DNA double determine towardsthe RNAPon the surface ,r, E. coli RNAP follows a helical path along the DNA template, duringtranscription researchersused a similar experimenlal geometry asdescribed Opticaltrap is near above. However, instead of using optical tweezers to trap the focusof the the bead, an iron magnet was used to hold a micron-sized laserbeam magnetic bead and stretch the tethered DNA template above RNAP a glass surface, i.e. magnetic [weezers. The magnetic bead Classmicroscope slide was labelled with small (20 nm) fluorescent beads, allowing the rotation of the large bead to be followed by viewing its magnified image from above by fluorescence microscopy ) AAeonified 6oad irnaoe Z lV \cf6lllllELl uuqw il rrq6u Magnifiednrot.t..nl. itug. (Fig 2). If the surface-immobilized RNAP followed the is observedfrom above showingrotation of magneticbead right-handed helical path of the DNA template, the bead should rotate once for every -10bp traversed(number of &#ffi @ s base pairs per helical turn of DNA). Indeed, the bead was observedto rotate clockwise at a rate < I DNA turn s-l during transcription, which was consistent with the linear velocity 20 nm beadsF Magnetic ffis@ s s (-10bps-1) measuredin the optical tweezersexperiments. bead The ability of the RNAP to rotate the bead, and by association ffi@& # * the DNA, also showed that it could produce torque just like a macrosconic molor. Classmicroscope slide @mss ffi Many bacterta are able to propel themselves through their

.a | | t. liquid environment using flagella. The roLary motor ,/\/\ Magnified fluorescence image ""*D" responslble for this propulsion is known as the bacterial is observedfrom aoove showingrotation of bead flagellar motor - it consists of -10 torque-generating umts arranged like a statlc bearing around the rotor shaft and ilrunffireanchored in the bacterial cell wall. The flagel1armotor can Truncated be fuelled by either proton-motive force or sodium-motive flagellar force, whereby the inward flux of ions acrossthe cytoplasmrc fragment roruHHffi membrane, and down an eiectrochemrcal gradient, drives rotation. The flagel1armotor should undergo discrete mech- ffiwruffiffilanical steps; however, its high speed and predlcted small lmagesare sampled at a step size had prevented the resolution of these steps.Using a i Classmicroscope slide rate of 2,400 framess-1 ;...... 1 bacterialstrain expressinga'slower' sodium-driven chimaeric flagellar motor, and high-speed video recording of a 200 nm &, i t::.',,i :, Determinationof the ability of RNAP to do work by watching it pull fluorescent bead attached to an intrinsica$ sticky flagellar on a DNA molecule(1 ), measurementof the abilityof RNAPto rotatea DNA filament (Fig. 3), researchershave been able to visualize indi- molecule and thus tracl

110 m icrobiolo gy today ;:iirli i-;iii F ;.

only one copy, which further reduces r ChristophG. Baumann the probability of a collision. These ; Lecturerin MolecularBiophysics, collisions are driven by thermal forces, :i^ Departmentof Biology(Area 10), which are random or stochasticevents. Fluorescencepeaks from i Universityof York,York YOlO 5YW, As a result, gene singleCFP molecules expression can often i Ul( (t"01904 328 828',* cb17@ be random too. h : york.ac.ul<) "6 The expression level c of a protein (J Cellular {r:*rt"**r P is usually determined by averaging {*a*|ftfr .g autofluorescence c a population of cells, which masks : Abbondanzieri, E.A., Greenleaf, WJ., \\ I its random j €(.) nature. By combining Shaeviz,J.W, Landick, R. & Block, \ (2005). I 1 laser-induced fluorescence and cell i S.M. Direct observation of t. microscopf (Fig. 4),the production of : base-pairstepping by RNA polymerase. U a fluorescent protein (GFP) localized 1 Nature438,460-465. t on lhe inner bacterial membrane could i Harada,Y., Ohara, O., Takatsuki, A., Position be followed in real-time in individual : Itoh, H., Shimamoto, N. & Kinosita, cells. Single-molecule detection of X. (ZOOt). Direct observarionof i A l:i1t,4. Using laser-inducedfluorescence fluorescent proteins in live bacteria, i DNA rotation during transcnption by and cell microscopyto follow the Eschenchiacoii RNA pol1'rnerase. productionof a fluorescentprotein (CFP) cells removes the aforementionec- : Nature localizedon the inner bacterialmembrane ensemble averaging. The GFP gene t 409.i13-115. 1 in realtime. C. Baumann,based on an was under the control of a repressed , Sowa,Y., Rowe, A.D., Leake, M.C., image in a Further reading article Iac promoter, thus protein production ; Yakushi, T., Homma, M.,Ishijima, A. 6r per revolution is consistent with the represented leaky gene expression. i Berry R.M. (2005). Direct observation number of FliG molecules thought to This wolk showed that each gene ; of steps in rotation of the bacterial | exist in the torque-generating rotor. expression event, or mRNA molecule, flage\Ia, motor. Nature 437, 916-919. This work begins to shed light on how was translated to peld approximately i Wang, M.D., Schnitzer,MJ., Yin, H., electrochemicalgradients are coupled four protein molecules per molecule of ; Landick, R., Gelles,J. & Block, S.M. to directedmotion and highlights how mRNA. By allowing low-copy-number i (1998). Force and velocity measured recent technological advances enable proteins to be imaged in real time, j for single molecules of RNA poll'rnerase. new milestonesin the single-molecule this approach allows the effects of : Science282,902-907 . l field to be achieved. stochasticityon fundamental biological j Yin, H., Wang, M.D., Svoboda,K., processesto be investigateddirectly i Landick, R., Block, S.M. 6s Gelles,J. 1;?j't: C*n s *'x.{}r"#*%i{} {1';Jt i (1995). Tianscription againsran applied ;} singn*-r.*Enfuv*1 Tvttts { * {}{ {3 "1.}t: q':,tr.:i i force. Science270.1653-1657 . All biologicalevents are dependent on The last decadehas seengreat advances , Yu,J.,Xiao,J., Ren, X., Lao, K. & the collision of one or more molecules. in the areas of single-molecule detec- : Xie, X.S. (2006). Probinggene The expressionlevel of a protein in a tion and manipulation. Theseadvances i expressionin live cells,one protein cell is dependenton severalcollisional will continue as new technologies r molecule at a time. Science3ll, complexesleading to'active' complexes, from the physical and chemical sci- I 1600-1603. e.g.one or more transcriptional acti- ences are applied in the biological vators binding to RNAP and the sciences.Such developmentswiil allow promoterDNA, and a ribosomebinding greater temporai and spatial resolution and translating the RNA transcript. In at both the single-molecule and single- addition,most genesin bacteriaexist in cell levels.

microbiologytoday ;l*sJ i)* Cor^tyou soetho llght?

hile recent advances in imaging technol- very expensive and rely upon bright lasers for illumination. ogies have had a dramatic impact upon Imaging multiple wavelengths invariably means using multi- 'widefield' biology, the technical challenges and ple lasers,further raising prices. Tiaditional, fluot- baffling array of options have tended to escencemicroscopy, on the other hand is versatile, affordable restrict these technologies to a limited and now sensitive enough to deliver top quaiity imaging. lt number of cogniscenti.This is particularly works by bathing samples in light of a specific wavelength true for imaging microbes where low intensity signals have that is selectedby the use of dedicated fiiters from a universal conspired with the need for considerable magnifi.calion to white light source.The secre[is to ensure that as much of the present a formidable challenge.However, some straightforward light that ieaves the specimen gels to the camera as possible. changesto standardfluorescence microscopes can now satisfy this obsessivedesire to maximize sensitivity and resolution. Enhancingan existingwidefield system "l Lighl p;tf i' []rilvi$i{}il lmagingplatform: spinning, scanning - The most easilyjustified modification to an existingwidefield how convolutedcan it be? system is to upgrade the light source from the traditional Laser scanning confocal microscopes (LSCMs) have a mercury bulbs to a tungsten/xenon system.As well as being reputation as benchmark instruments for high-resolution brighter than mercury bulbs, tungsten/xenon systems are imaging. Howevet, they require very bright samples and much cheaperto mn (10.35 per hour vs. 1I.20 per hour). most microbiologists have no need to take advantage of Thus, they not only boost sensitivity, but pay for themseives their ability to illuminale a constrained slice in thick sample. over a 2- to 3-year period, and then save money after that. Although LSCMs are not ideal for imaging bugs, a second A final perk is that they overcome [he common pitfall of type of confocal microscope, lhe spinning disk microscope uneven illumination that has plagued the use of mercury (SDM), is. Instead of scanning consecutivepoints in a series sources. The xenon/tungsten systems use a fibre optic cable of lines across a field to build up an image line by line that that scrambles the image of the bulb to deliver an even is the hallmark of LSCM, SDMs simultaneously monitor illumination that is invaluable for quantification. 356 points which reduces off-site bleaching and combines wrth further lechnical advanLagesto give arguably the best J" I-ightg:*t i"r: tr*"n*rnlEsiern temporal and spatial resolution available.However, SDMs are When passing through, or bouncing off, an optical element some light is lost, eroding both the intensity and quality

A Microscope lenses. David Parker/ SciencePhoto Library of the image. While the optical elements in microscope

112 microbiologytoday aug 05 Amazing findings can be made using fluorescence micros copy, buthow do you choose the right equipment? lain Hagan, Agnes Gralfertand Steve Bagley give some practical help. Sir-npleond offordob stepsto exploit reoentodvonoes i fluoresoenoeir-nog ng bodies have been reduced to a mini- mum, the demands of certain disci- plines mean that objective lenses can contain a considerable array of additional elemenrs. Pathologisrs and neurobiologists often want to see high- power magnification lmages ol entire fields that are free from distortion and in sharp focus from one side to the other. This technically challenging demand is met by the inclusion of correcting elements to counteract dis- tortions at the edge of a field of view. However, these extra elements reduce transmission.This cost is too great for microbiologists who would happily toleratedistortion at the periphery of a field that is often tens of cells wide, as the benefit of neglecting this correcrion is enhanced transmission. In other words because commercial suppliers offera range of lens types for a rangeof

) Fig.1. A cartoon showing the two optionsfor filtrationof light in a widefield excitationfi lter wheers (b) microscope system. (a) Most systems use ' filtercubes that sit between the objective Tr- and the eyepiece;however, the use of filter Emission wheels(b) enablesrapid switching from filterwheel one channel to another. Steve Bagley T:*1ffi

.{# Spectralprofile of CFP applications it is vital to know what rype of lens you are using. Does it have a lot of correction or maximal transmission? The key term is rhe Numerical Aperrure (NA) of an object_ ive. The higher rhe NA, the higher rhe rransmission. euite fortuitously for microbiologists, a form of microscopy cailed total internal reflection microscopy (TIRF) that demands the highest NA possible has recenrly gained in populariry, inducing every commercial supplier to produce objectives with extremely high NAs. The catch is an exrremely - Excitation sharlow - - Emission depth of field however, the size of microbes comes to our rescue again as depth of field is rarely a problem for a microbiologist. A furrher carch is that you are likelv to be told by the sales representative that these lenses are only useful for TIRF and you should nor try them! However, persist! Demand a loan for a trial run! A switch from a I00x Longpass CFP filter 1.3 to a 100 x 1.45 doubled the intensiry of our yeasrimages. Many microscopes have optional internal magnification lenses on sliders or wheels that can provide further magni- fication berween the objective and the eyepiece/camera. s Beware, these lenses can severely erode image quality and c shouid be o used with utmost caution. .!3 E 3. l-iEhfrpaith; \{d;}\r#1flr"}Sths*imcfi*n c - (d Excitor Fluorochromes are excited at one wavelength and emit light - Dichroic at a second, lower energy (longer) wavelength. * Emitter Thus paired excitation and emission filters that select the appropriate wavelengths for a particular fluorochrome lie ar the heart of any widefield microscope. Most fluorescence microscopes have filters paired up, either side of an appropriate (dichroic) mirror to reflect and transmit Narrowpass CFP filter the appropriate waverengths in specific blocks between the light source objective and eyeprece/camera(Fig. 1a).Aiternatively, and more expensively, filters can be mounted on independent filter wheels to enable more rapid changesin wavelengths (50 ms vs 900-2000 ms for changes of internal filters) and to isolate the movrng parts from the body of the microscope to minimize vibration (Fig. lb). Whichever sysrem is available it is imporranr ro understand the range of filters at your disposal. - Excitor Fluorophore emission spectragenerally have astrong peak - Dichroic that trails off towards rhe red * Emitter end of the spectrum (GFp in Fig.2a). To distinguish berween rhe signals from two different fluorochromes in a single specimen, two combinations of excitation and 400 500 600 700 800 emission filters are required to select the Wavelength (nm) appropriate portion of the spectrum to excite and observe one fluorochrome while excluding those that would excite/ L Fig.2.Filtration of CFPemission by long and narrowband pass observe the signal from the other. Tvo issues arise here. The filters.(a) The excitation (blue) and emission(green) spectra of CFp. more resffictive the filtering, the more Thegreen bar indicates the breadthof ffuorescenceemission that rs sophisticated is the fullysampledbythe longpass (b) but notthe narrowpass filter (c). coating on the glass needed to generate the filter. The more SteveBagley complex the coating, rhe less light is transmitted. Second, the selection of the narrow bands of the spectrum that are ) Fig.3. Labellingliving 5. pombecells with two ffuorescentlectins required to see multiple facilitatesthe ultimatelive cell imaging control - threedistinct cell wavelengths as separate signals in typesimaged alongside each other. Red, celltype 1;green, celltype one sample excludes the long tail in the emission spectrum 2; black,cell type 3; blue,chromatin (Hoescht 33342).Asnes 1rallert of the fluorochrome and so effectively rhrows away up to

114 microbiology todayau6 0d I

20 o/oof the signal from the sample. Therefore, if only one fluorochrome is Somestraightforward changes to 'long to be imaged, pass'filter sets (Fig. I 2b), that need less coating and lack an standard fl uorescencemtcrosco pes can upper cut off, enabling the full tail of I the spectrum to be captured, give much now satisfythe obsessive desrre to better signals than the restricted and heavily coated'narrow band pass'filters maxim rze sensitivitV and resolution that permit selective imaging of differ- ent fluorochromes in a single sample that are transforming the quality of lies outside the box. It can therefore 'heaL (Fig. 2c). If you are just interestedin cameras in high-street stores are also act as a sink' that reduces the GFPin an experimerLL,a long passfilter ffansforming microscopy For microbio. temperature in the one place where set will boost intensity by up by 20ok. ogists, the key issue is the quantum you do not want it changed - the field Furthermore, the technology used to efficiency of a CCD chip - iiteraily, how of view. This problem can be overcome generatefilters has been revolutionized much light arriving at the surface will with accurate and highly versatile in recent years so that simply replacing be converted into an electricalread out. commercial systems that control the old filter sets with newer aiternatives A technology in which light signals are temperature of the coverslip surface (splutter-coatedfilters) can increasethe converled into a stream of electrons has (for example, by heating a rransparent o/o. brightness by 20-30 pushed quantum effi.cienciesof chips to coating of metal) and the temperature One aspect of light sampling that is greaterheights. Switching to an'electron of the objective with a heating collar. A often overlooked is the type of dichroic multiplying CCD' camera has revealeo number of micro-perfusion chambers mirror used.Just like filters, the greater signals that we could not detect with can be used to maintain nutrient sup- the demands placed upon the specificity the previous generation of chips. plies or deliver drugs or toxins on cue. of the mirror, the more sophisticated Cells can be mounted on a range of are the coatings,so less light is Lrans- li. {}i: Ii ru:,,i{s;i ru t}i# r:-,;ii"ri:i"riXl transparent gas-permeable matrices mitted.Thus, the selectionofadedicated :;t;ryi*g ,l-iii;r: fo ensure [he correcL aerobtc/gaseous dichroic for a particularwavelength can Environmental control can be as criti- conditions are maintained over extend- alsoconsid erably boost transmission. cal as the optimization of the light path. ed periods. For example, encasing the stage in a 4. l]*te,:i:fiqltt " t:*tr"]:ii.ir,it heated Perspexbox might not be suffici- i.r 'rl' ll t. ..,',:iiiir),l.J.ti!1i Camerastake us away from this articles ent to give accuratetemperature control. Microbial cell walls can be exploired 'easily theme of affordable' adaptation. The metal of the objective lens is con- in a variety of ways to assist imaging. However, changes in CCD technology nected to the microscope bodv that Coating coverslips in ligands that bind

microbiologytoday ;r.r-ri:.*fr cell-wall componen$, such as poly- r--lysine or lectins, is a simple way of ensuring a sample stays put during image capture. This is particularly EEEE important if medium has to be changed via perfusion. Another simple trick is the selective labelling of one cell type in a single field of view with a fluoro- EEEE chrome before they are mounted side by side in the samefield. Thus, a mutant strain can be observed in exactly the same conditions as the wild-type con- EEEE trol (Fig. 3). ls it worth it? A modest equipment grant of around ETEE 112,500 can transform an existing microscope system for imaging GFP to deliver some top notch images (light source,{,3, 000-4, 000 ; spectalized EIEE objective, f,5,000-7,000; dedicated filter/dichroic set, 1800). A quick trip to another lab that has the technol- ogy in place can determine just how ETEE great a difference can be made and whether it will be of any use. Invari- ably the gains obtained with these modest adjustments eventually provide ETNE justification to grantingbodies for a new camera and more sophisticated image processing,leading to the abilrty to visu- alize three or more proteins for extend- TIIE ed periods (Fig. a). There has never been a better time to dispel the folklore surrounding imaging and have a go. lain Haganl,Agnes Crallertl TIIT & SteveBagley2 ICRUl

A supplementwith further tips to negotiate imagingare linhed to this article on the Micro- TIII biology Todayw ebsite at ww w.s gm. ac.uh LFig.4. Three colour imagesof dividing S. pombe cells in which the centromeresare marl

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& Portraitof HenryBaker. A stippleengraving by William enry Baker(1698-1774) havebeen mistahen, let not vanity seduce Nutterpublished in 1812and taken from an earlier was oil paintingby WilliamThomson. Baker was variously a typical l8th cen- you to persistin your mistahe.' describedas upright, benevolent, patient, homely, quietly tury polymath: natural This is Bakers cautionary advice in - spol

118 microbiology todayaug *{{ t"TfIT ffi FfrICROSC()PE IVIade Eafy : ' :tgt i': rx-' i. Eig$^rr:'t:n .,: t"ft11ltlg31 tfsnfr,u#"r,o,1""y, es defirc to fe.uch into.tlrc _1,I/o1c n i n s of tt.a-J6lia*t"C.iir;i_ 'i"l tno- tnc/ are flot zcguaintedwithaptits. i^i N *t6 i ir. i i i-i-]iii"i.ri_l i: Frdt' -' trdrccfionr l,o* to #$* ;,' s;ffi'#' --'" 4V,-;#{i'ja :#r6ry'&*Hg4*. o m bc o6&rced" vicwjns'thqh. #"#:?l:-;L*Hff tiil1F" f#fl ** ffi Witb u&lr.rl Rc ,ffi RichardBurns,

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seenin rain,ditch and pond waterare relativity ('Our ideasof matter,space, and 1000 copies of The MicroscopeMade illustratedand there are descriptions duration are meerlycomparativ e'). Easy were printed and sold so rapidly of blood, muscles, bones and nerves, Towards the end of the book Baker that it was reprintedin April 17+3. qemen the lorrc,e fleas znd cniderc. (bee comparesrr the beauty of natural Soon other microscopical studies were and how to kill insects with tobacco sting, silkworm web) and man-made published, but this book had opened oil and mercury. One chapter contains objects (lace, works of art). To him rrn fhe rnqr]rot a^ -^^rag-Daq L^^ or ^f oDservatrons^r-- rangtng the artefacts come a distant second. Elevenyears later. in Employm(nt 'Our Jlr from the behaviour of ants to snow- fnest miniature paintings appear the Microscope,Baker humbly advises flakes.All come tliterally) under the before this instrument as meer dawbings, criticalassessmentof the book'scontents microscope. plaisteredon with a trowel, and entirely and further study, and declaresrhat his Rrl,er rcfer< frenrrenllv,.",,/." tn thc e:srlier void of beauty.' target audience is wide ('many of both drscoveriesof Hooke, Swammerdam, Leeuwenhoekand others.But he goes : ': further and exnresseshis views on ti), Title page of TheMicroscope Made Easy(2nd edn, 1743) A total of six Englisheditions were publishedfrom 1742to 1785 as well as Dutch, Frenchand Cerman translations. manv of the conrroversialissues of "----l The book sold for 5 shillings(about f30 today)and Bakerreceived the equivalentof the duy For example. spontaneous approximatelyf5,000 for the first edition and half that for subsequent editions. generationrs ('Nothing ' dismissed seems ,li :,;, The double reffectingmicroscope. This is describedby Bakeras an improvement nlw morecontrary to reason... thatdead on John Marshall'sturn of the centurygreat double microscopemade initiallyby Edmund corrupting matter, and blind uncertain Culpeperin the 1720s and then by EdwardScarlet in the late 173Os.The body is supported by three brasspillars mounted on a wooden base.There are five objective chance,should create living animals.'). lensesthat can be screwed to the nose piece (g). The brassplate (O) fits onto the fixed plate (L) and contains but prelormationgets the thumbs up holes to carry specimens;ivory discsare used as the bacl

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sexeswhl have not had the advantageof In 1740 Henry's influentral friends l* a learned education'). There is much elected him a Fellow of the Society I i informatron on insects,polyps. rotifers, of Antiquaries and, the foliowi.ng year, I nema[odes and fungal spores as well to the Royal Societ;r His citation reads 'A as detailed accounts of Baker's own GentlemanweII yerse din Math"ematicks ^-i^^,^r-i---,,,r,es )LL-lLl[LJ vrof inorsanicand andN aturaI h.nowledge, p ar ticularly emin- P4lll)(clt\IIIS "'v'bd'rMrr ^-^^^:^ -^1,- ^^) LJrSdrrrL )drL) arrLl how these1ed him ent for his great SkiII and hoppy Success to suggest mechanical improvements in teachingperslns born deaf and con- lo rhe microsconefThe maior..'*.J"."t/..., ontical sequently Dumb to Speak ftaving im- advancethe adontion of the achromatic proved upon that great Invention of the lens to reduce distortron, came much late famous Dr WaIILs)Author of a very later.] In fact, crystals were Baker's beauttfullPoem called the Universe,with first scientific love ('their vanety and mdny Cunous Nofes regarding llatw'al beautyn0 wordsor langudgecan possibly History,and onewho hath communicated "'r''""e,xnre.ss')and for this he received the Someusefull papers to the Royal Society, Cnnlevonld medal nrrttinphim on a beingdesirous to becomeaMember oJthe 6"'",y*..'''6 'giants': list of lBth century Franklin, Same, is recommendedby us os a Can-

I I Tr I HrresllP\- 2nc volla. didatew eII deserving th at hortour.' The six

nronosersinclrrded the heavrrweipht>,./,,-.b.^. Hans Sloane(President), Martrn Folkes (Sloanes successor) and Cromwell 'day Bakers lucrative job' was as a Mortimer (BiologicalSecretary). sneech theranist",.'-r'"' and' in 1724 he was BakersB0 nlrs communicationsto teachrngin Stoke Newington when its the.^^-''"-/-,"'-^''-/ Roval Societvbetween 1740 and most famous resident, Daniel Defoe, 1769 are a mixture of the good, the hrd anrl the rrolrr Thcrr are rr.roqtl invited him to cal1.Despite a 40 y?ar -b',/' ' "-t - -' Y

q5rnc'e urrrLclif{erence r L rhe two hit it off and short papers and letters.Many appear Baker became a regular visitor. Soon, trivral and reports about the weather, however, the highlight was afternoon avalanchesand minor earthquakes are tea with Defoes daughters and Henry common. One of the few substantial inn nf the Snr-ietr"5 found himself falling in love wlth the v-v''naners is a descrint volrnsest.-/"-''b'"-'"-r'''*.Sonhia but it wasalmost four collection of 26 Leeuwenhoek micro- years before increasingly acrimonious --"r--'scones Baker calcrrlates their maonrly- " "'-^' "'*b'^"- negotiations with Defoe were resolved ing power (the best was about x160) anrlthe nairwere married. and concludes that by using these il John Cuff'snew double microscope,made in 1744 During this time, Baker launched Leeuwenhoekcould not possibly have 'when following advice from Henry Bal

120 microbiologytoday --.-'

earlier, had cut up fresh water polyps (Hydra wlgans) and In his will he left 1500 to the Royal Society (in addirion found that the parts regrew to form entire animals. This to the Bakerian lecture bequest of f,100), but mosr of his news excited the scientific establishment, because of the considerable fortune went to his grandson, Wlliam The debate about the distinction between animals and plants Bakerian lecture ('for an oration or discourseto be spo'henor (only plants grow from'cuttings'but only ammals move and read yearly by someone of the Fellowsof the Societyon such eat worms), as well as the natural philosophers who were a part of natural history or expenmental philosopLty')was 'soul' puzzled as to how the of the polyp was distributed firct oirrenin 177\. Early speakersincluded Cavallo, Davy among the progen;r Baker was amazed by what he saw as he and Faraday and many of the greatestscientists of the day, choppedLhe hydra into pieces. including Rutherford, Herschei, Fox Talbot, Maxwell and In the Iate 17 40s Baker'sinterests turned to electricity (the Hoyle, have delivered the lecture. hot topic of the day) and he reported experiments that were Would Henry Baker be remembered if not for the thought to show its beneficial medical qualities. eponymous lecture and footnotes in the many Daniel Defoe biographies? There is no doubt that The MicroscopeMade " "i,:i Tlz t:: ilr i +:r::,::i ;,: ", l"* t: t::.1"1* A:l Easywas widely read not only by natural historians, but also Henry Baker's most prolific correspondent was William writers, poets and thinkers of the day. Baker's purpose in Arderon, an obsessivecollector of objects and obscure facts writing the book was to popularize the use of the microscope who wrote about what he had seenand heard. In return Baker and instill in others a curiosity for objectspreviously invisible kept his pen pal up to date with events at the Royal Society to the naked eye. These aims were achieved. Henry Baker 'a and life in London, and describes his editing activities, of was once described as philosopherof little things',which which two are notewofthy can be interpreted in two ways: a man who thought deeply In the mid 1740s, the surviving plates for Robert Hookes and wrote extensivelyabout the minute objects he examined classic Micrographia were cleaned and the missing ones under the microscope or one who spent his life collecting 'overloohing re-cut. Baker tells Arderon that he is: the press and explaining essentially trivlal observations. The former which a little worh of mine has just now passedthrough. I caII definition is the accurateone. it MicrographiaRestuarata or the Copper PlatesoJ Dr Hoole's wonderful discovenesby the microscopereprinted and fully RichardC. Burns explained.'Baker says that he has asked the booksellers to Schoolof Landand Food Sciences, University of price it as iow as possible to attract customers and convert Queensland,Brisbane 4072, Australia (t. +51 7 3365 them to the wonders of microscopy. 2509;*: r. bu [email protected]) In August 1749 Baker informs Arderon that he is involved in editing Benjamin Wilkes' The EnglishMoths and ButterJlies ,i..., ts 1i,: and pays Wilkes a wonderful backhanded compliment: 'indeJatigable Baker, H. & others (1740-1768). Paperspublished in the in his obseruations,and fatthful in minitigg down PhilosophicalTransactions oJ the RoyalSociety . JSTOR- The every p articular, but for want of learningquite incap able of w nting Scholarly J ournal Archive (wwwj stor.org). abooh.'Butthere is no mention of Baker'sinvolvement when Potter, G. (1932). Henry (1698-1774). this beautifully illustrated book was published, although Baker,FRS ModPhilol 29,310-32r somelines on butterflies from The Universeare included. George Adams was a prominent instrument maker Turner,G. LE. (1974).Henry Baker, FRS: founder of the who could see what The MicroscopeMade Easy was doing Bakerianlecture. Nofes Rec Roy Soc 29, 53-79. for the sales of Cuffs instruments and in 1746 published Victoriaand Albert MuseumForster Manuscripts. The Micrographialllustratain which he describesthe merits of his collectionincludes four volumesof Baker'scorrespondence wrth own microscopes.However, Adams had plagiarized sections WilliamArderon and his autobtographicalmemoranda. of The MicroscopeMade Easy and Baker was incensed. He Woodruff,L.L. (1918).Baker on themicroscope and rhe wrote to his friends warning them of the'notorious robbery' polype.Sci Mon 7, 212-226. 'an by ignorant but impudentJellow' and urging them not to buy the book.

"rI : i il {:l {:il"i {-J Henry Baker died ln November 1774 and was buried alongsidehis wrfe in the churchyard at St Mary le Strand.

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microbiologytoday SchoolsMembership costs only f.10 ayear.Benefits include MicrobiologyToday, advance copies of new teachingresources anddiscounted fees on SCMINSET courses. To join see www.sgm.ac. u k/mem bersh ip. En q u i ries : education @sgm .ac. u k or go to www.microbiologyonline.org.ukfor full details of resourcesand activities. *r,r*ffir t*ffi rT3v*rmffi**wffi

Usinga microscopein school to lookat microbescan moulds and even yeasts,the low power objective lens (x10) is often but isnot as difficult as it seemsif adequate,but also necessaryfor bevery rewarding locating and centering on an area of interest before turning to the youfollow John Grainger's handy hints. high power objective lens (x40). Without altering the focus, turn to the high power lens and then The setting up of a microscope is fine1yre-focus. a basic skill that is rarely mastered Use the oil immersion objective in schools. Only when it is done lens for examining stained properly can the smalier end of the preparations of bacteria. Put one diversity of life be fully appreciated drop of immersion oil onto the and microscopy be put to many other preparation;a coverslipis nol, uses,such as aiding identification, required. Remove the slide and measuring growth and checking for wipe the oil immersion lens , contamination. The magnifying power clean after use. of a microscope is important but, particularly when looking at microbes, More detailed information on remember that the amount of detail microscopy,including observing seenis determined by its resolving and preparing mounts for observing power. The sub-stagecondenser different types of micro-organisms, plays a crucial role in achieving making smears and staining good resolu[ion. procedures is available in Basic Practical Microbiolo g : A Manual F4|nts (email [email protected] Adjust the iris diaphragm to obtain a copy). achieveoptimum balancebetween defi"nitionand glare. Do not control JohnCrainger is Chairman of light intensity by moving the sub- theMicrobiology in Schools stagecondenser, the position of AdvisoryCommittee (MISAC) and which should be to focus the light co-deliversSCM's basic oractical on the specimen. Re-adjust the iris microbiologycourses for teachers diaphragm for each objective lens. andtechnicians. He can be

& Youngschoolgirl using a microscope, For looking at wet mounls of contactedvia SCM HQ. RichardBailey / SciencePhoto Library living specimens of protozoa , aIgae,

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SCMaims to promotemicrobiology education itsbroadest sense. VrJ{::::}ti*r}{::}Z NationalScience Weel< (NSW) provides anideal opportunity to raisethe profile of our subject. 3ffis/ T,t"*7%.,,2mw describes {:::}ry{:ry""'Mf: someevents that were supported by u 'tr: grantfrom the Society'sPUS Fu nd. /3r*:**;{:i{,*ffir1 1;.111 - %7,* rl r-it rz1; Ii gErt rJ{'t {:: The three Ps Perkins, Porter &rr|t,1,;:i T.'t'ifi5{} {-ti l::1;. "u r.sr 1r:i r:.stt* *il and Pennington - also helped to raise nzir:rr:ls*y, the profile of microbiology at the NSW ProfessorKen Killham delivered the ScienceDiscovery Day. The excellent NSW lecture on this topic at Aberdeen venue for this event was Satrosphere, Universityon 16 March 2006. He Aberdeens hands-onscience centre, provided a fascinatinginsight into the and it was organizedby the Aberdeen tiny organismsthat live underground. Branch of the British Association for (BA). The event was attended by Higher the Advancement of Science and Advanced Hlgher Biology pupils The event was only made possible from local schools. It also featured a by the generosityof BP who covered competition with GIANT Microbes" admissions chargesfor 800+ visitors. as prrzes.These are educational cuddly At this lamily-orientatedday I toys modelled on viruses and bacteria. delivered a workshop calledkees, - Ken explored The Good,The Bad & plants and microbes gettingto the root TheUgIy of soil mlcrobes. The Good oJthe issue.Vlsitors found ou[ about graduate from Aberdeen Universit;r focusedon a range of beneficial soil microbesthrough a rangeof In the lBBOs Ogston discovered and plant-microbial associationssuch hands-on activities. They discovered worked on Staph.du.reus and researched asmycorrhizal systemsfor nutrient how tiny organismsin soil help trees the causeof hospital infections. and plants grow and lnvestigated captureand the use of microbes to Many thanks to SGM for funding both beneficial root-microbe interactions. cleanup persistent organic pollutants events and Aberdeen University staff ExampleBoi friendly mycorrhizal in soil. TheBad investigatedthe deadly (too many to mention individually) for fungi from the roots of orchids and a sromachbug E. coli Ol57 .H7 and their support in raisingthe profi1eof pine tree were also its presencein Scottish cattle, as well on display Drag microbiology during NSW 2006. as the tery bad endospores of the soil and drop computer exerciseswere bioterrorism bu g Bacillus anthr acis . very popular with visitors of all ages. JoyPerkins TheUgly covered devastating plant PeteJeffels from the Learning Schoolof MedicalSciences, pathogens,including winter wheat Technology Unit at the University used Universityof Aberdeen,Aberdeen root rot. At the end of the talk the Flash Macromedia software to design AB252ZD (* j.perki ns@abd n.ac. uk) audiencewas reminded that the soil mushroom-basedactivit ies. If you aregoing out into the community sustainsthe planet - providing our In the Satrospherecafe, Professors to promotemicrobiologt, a grant food and fibre, regulating our water Andy Porter and Hugh Pennington from the SGM'sPUS Fund can provide up and air quality, supporting trees for contributed to the RealLive Science to Ll,0 00. Se e w ww.s gm. ac.uW gr ants timber and fuel, and cleaning up most programme. As visitors relaxed and detailsof the scheme.We of our pollution. The lecture was also enjoyeda bite to eat,scientists gave for further can alsosupply ideas activities pepperedwith interesting facts, for short presentations.Andy described for and providegoodies to give away,as well example,in a single hectare of land the human immune system and how as tell you what is safeto do! Contact thebiomass of microbes equatesto 30 it fightsdiseases. Hugh. usingthe [email protected] sheeplProfessor Killhams enthusiasm memorable title Bird'Jlu! MRSA!Wd re wasinfectious and the event was a all doomed!?,provided a lively account 'Real A ProfessorHugh Penningtonat the hugesuccess with visitingpupils. of Sir AlexanderOgston. a famous LiveScience' initiative.

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%t"Er*rzr"eir: S*h**E * www" SGM sponsored the l8th MISAC competition for secondary schools. This year's sr.i * r"s*ei ms t ?tn"s n*l. rs rg topic was chosen becauseof the serious problems causedin hospitals by MRSA (methicillin-resistant Staphylococcusaureus) and their extensivecoverage by the This is a new Europeanjournal to media. Therefore, it was no surprise that the competition to write a newspaper promoLeinspiring scienceteaching. feature on a hospital outbreak of MRSA proved to be very popular. It generated It covers the whole range of science, nearly 700 entries and involved more than 800 students from 105 schools and highlighting the best in teaching and collegesdrawn from England, Wales, Scotland and Northern lreland. Pleasingly, cutting edge research.It is availablein this year there were more entries than usual from the GCSE Group, with print in English and online in several numbers still holding up well in the I1-14 age range. different European languages. The judges looked particularly for attention to the guidance grven to entrants on To receive an aleil when an issue is the writing of a news story including preparlng the headline to catch the reader's published, send an email with the attention, structuring the story to maintain interest while conveying essential subject Subscnbeto Sciencein Schoolto information, and the appropriate use of pictures, diagrams and scientific terms. [email protected] Other important featureswere evidence of scientific merit, the use of entrants' own words rather than text downloaded from the web, and an appreciation of Where** m*dieFs"":*st*m* the importance of bringing out human and local interest. Tr*mr SueAssinder andJanet Hurst, representingSGM, joined the Chairman and ABPI has put together this new other members of MISAC for the judging at Marlborough House. This year the resource for primary schools, designed judging panel also benefited from the expertise of Alexandra Blair, Education to be used with interactive white Correspondent on TheTimesnewspaper. Many entries impressed the judges with boards and computers. It is in three their high quality, but unfortunately a notable proportion were excluded from pafts: consideration becausethey did not adhere to the competition rules, particularly where do medicines come from? - by using the format of a factsheetinstead of that of a newspaper anicle. Powerpoint presentalion of stories The winner of the II-L4 age group was Eleanor Tayler of the Abbey School, for KSI and KS2 with follow-up Reading, Emma Pascallof Durham High School came first in the GCSE group. activities Further details of the winners are avatlable on the web (www.microbiologyonline. how do we get medicines in our org.uk/misac) and a selection of the entries will be on display on the SGM stand bodies?- a seriesof animations at the ASE annual meeting at the University of Birmingham (4-6 January 2007). posters - A2 sizeto be used at Each school entering the competition received a pack of microbiology teaching different points in the primary resourcesand every student was sent a certifrcateof entry curriculum. Next year'scompetition on Salmonella:fromfarm toJorh, will be sponsored by the Seewww. abpischools.org.uk Society for Applied Microbiology and an entry form will be downloadable from for details and to download web the MISAC website (seeabove) in September.MISAC is grateful to all its sponsors versions. for enabling the competition to take place each year.

€ TheMISAC judgingpanel at 5CM HQ in April 2006. Fromleft to right:Peter Fry, JohnTranter, 5ueAssinder, JanetHurst, John Craingerand AlexandraBlair.

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Cradlineaims to informand entertain members in theearly stagesof theircareer in microbiology. lf you haveany news or stories,or wouldlil

It may be the height of summer, but for some UK and Eire based postgradsthe rapidly approaching aulumn brings thesis submission deadlines and, of course, the viva' Some of you may have pushed thoughts of the final hurdle to the back of your minds - but don't leave them there too long. Despite the great mystique that sti1lsurrounds the viva (who hasn't heard tall tales of marathon 5 hour ordeals?)rnformation is availableto PhD candidatesfrom individual universities, websiles and books, not lo mention supervisors.To surl you off, we offer a few words of generaladvice. Obviously, for specifi'cinformation about PhD examining procedures,you must check out your own institution's regulations and talk to your supen'nsorto find out what to expect on the big day. The viva is the fi.nal stage of PhD training and is an opportunity for students to defend their thesis and show that they really can be independent researchersin their field. Ir is important to note that there is no standard format for a viva. Each PhD and each examiner is unique so styles do vary enormously However, all vivas aim to answer a number of common queslions: Did you do the work yourself? Are you capableof original thinking i.e. did you have your own ideas during the research? Did you write the thesisYourself? Can you identify the value of your own work and put it into context of the wider field of research?

Theexaminers The examining panel usually consists of an exlernal examiner (chosen carefuily by the superv'rsor- somelimes with input from the candidate) and an examiner from within your departmenl who has not had any involvement with your project. Some universities also require an independent chair to be present and the supervisor may sometimesattend (by invitation) to (usually silently!) support the candidate. Before the viva, the examiners mus[ wrile reporls on the thesisaccording to university guidelines or using a pro- forma. Although the examiners may have formed an opinion of the research,this can be changed during the course of the viva. After the viva, it is usual for both examiners to have to complete a joint report/pro-forma. You might want to ask your supervisor to show you these forms [o seewhat the examinersare being asked to say about you.

microbiologytodaY Thequestions If you encounter aggressionor hostile If feasible,publish your work before - Examiners will ask a range of questions, try to work out why this writing the thesis it always helps questions designedto obtain is happening. Sometimesprobing to have beensubject to positive peer different kinds of information. Good questionson a weaker areaof your review. examiners should not be aggressive thesis can seem very intimidating. Keep reading the literature after but they will ask quite searching and Maybe your examiner is tD ng to make you submit your thesis, people do comprehensive questionswhich can you see another point of view. Perhaps publish all the time and you don't your examiner has an aggressive typically be about your Journey', the want to be caught on the hop. researchcontext, your methodology, speaking style. Stay calm and defend Work out how [o summarize your results and conclusions. your position rationally If necessary succinctly what you did, why you you should concedesome points or Journey questions give the examiner did it, what you found and why this agree to disagree.The chair and/or a feel for your personal voyage of is important. internal examiner are there to ensure discovery. Prepare a list of questions that are fair play Why did you choose this field of likely ro come up - rhen think Most vivas last between 1.5 and study? about your answers. 3 hours. At the end, it is common What was the most significant Look critically at your thesis and if practice for the candidate to leave the challenge you have faced in this you spot errors or weaknessesdon't room during the examiners' discussion. work? try to hide them, be ready to discuss This can be nerve-wracking and you them with your examiners. Which aspectsdid you enjoy the may prefer to spend this time alone, Don't dig yourself into a hole! Be most? although some people do line-up some honest and admit to not knowing If you could go back and do it all suppoftive compan)r Once called back answersto questions. again what would you do into the room there are a number of Recognizethe strengths of your differently? possibleoutcomes. Not all of theseare work too. Context questions test whether you available a[ every institution. Researchyour examiner - what can put your work into the wider PhD awarded- no corrections are their interests?Do they have any research,/societalcontext. (unusual but by no means biases?Bear these in mind during Which techniques do you wish you impossible) the viva. had been able to use? PhD awardedsubject to minor How would you apply your findings corrections(usually approved by to...? internal examiner within a set time Goodluch! Tell me more about the work of period), JaneWestwell X and Y and how it relatesto your PhD not awardedbut candidatemay ExternalRelations Offi ce findings in Figure 4.2 submita revisedthesis to both internal What are the implications of your and externalexaminers (sometimes Bob Rastall fi.ndingsto public health policy...? known as major corrections) Schoolof FoodBiosciences, Chalienging questions make you PhD not awardedbutcandidate Universityof Reading defend your position. must resubmita revisedthesis and also Do you really mean that...? undergoa secondviva Furtherreading I can think of at least three Recommendationthat alower degreeis How to surtiveyour viva (R. Murray, alternativeexplanations for Figure awarded 2004, OUB ISBN 0-335-2I284-0) offers 5.3 - why shouldyours be correct? Candidateadvised to reyisethesis and a detailed insight to the viva process including questioning styles and suggests Yourdata in Table6 contradictsthat submitfor a lower degree strategiesfor success. of Xxxxx et al. (2004) - who should No degreeawarded and no opportunig The doctoralexamination process: a. we believe? to submitJor lower degree handbooh f or students,examiner s and superttisors(P Tinkier 6l C. Jackson, 2004, Yourresponses Othertips for success OUB ISBN 0-335-213057) - information, In your answersyou must A successfulPhD candidate shows advice, real-life accounts and casestudies demons[ratethat you can put your excellencein both thesis presentation based on a review of the examining work into context and you must have and performance during the viva. process in the UK. justified critical opinions on the value There are a number of things you can of your work do to ensure your own success: 1 PeterCade / Cetty lmages

microbiologytoday x*g ii{i 129 Sciencewriter Meriel Jonestakes a lookat somerecent papersin SCMjournals which highlight new and exciting developmentsin microbiologicalresearch.

Rabiesin the air the few casesof rabies, including one of Dstl, Porton Down, have been testing a bat conservationistin the UK, where whether Lyssovirusaerosols can cause Johnson,N., Phillpotts,R. & Fooks, the route of infection has never been diseasein mice. They used strains A.R.(2006). Airborne transmission confidently identified. of both RABVand alsoEuropeanbat of lyssaviruses.J Med Microbiol55, lyssavirus2 GBLV-2), isolated from a One concern is whether lyssaviruses 785-790. Daubenton'sbat that causesa rabieslike can causeinfection from breathing disease.Some mice becameunwell after is rightly feared, but measures an aerosol.This could come from the Rabies either virus was inoculated into their reduce its incidence have been very large amount of bat urine and guano to nosesand aerosolsof RABV over a 100- within Europe, especially around bat colonies in cavesand other successful fold range were able to causeillness. in However, the Rabiesvirus pooriy ventilated spaces.The risk the'UK. This provided convincing evidence that (RABV) causestypical rabies is only would depend on how much virus was that the rabies virus can be transmitted by one member of the genus Lyssafirus. in the air and how well it survived, as these unusual routes. Others aiso causedisease in mammals, well as whether it could actually cause including fatal illness in people. These an infection after passingthrough the However, more encouragingly,the viruses are found in bats, although the surface of the respiratory tract. As a first mice that had breathed an aerosol of animals are unlikely to bite humans. step in evaluating any risk, researchers EBLV-2showed no signs of any illness, However, they are considered to be a from the UK at the Veterinary suggestingthat contact with the virus new public health risk, maybe linked to LaboratoriesAgency, Weybridge, and from bats Dosesa lesserrisk.

BSE- no prionsdetected in milk Miik is a difficult material to test for BSE.All tests require appropriate negativeand positive controls, but there is no Everest,5. J.,Thorne, L. T., Hawthorn, J. A. & others(2006). naturally infectious milk to use as a positive control. Milk is No abnormalprion proteindetected in the milk of cattle predominantly water, but the researcherswere certain that infectedwith the bovinespongiform encephalopathy agent. any prions would be within the very small amount of cow J 6en Virol87,2433-2441. t cells also present in milk. They concentratedthese cells for testsand used samplesspiked with authentic BSEprions to One niggling question about BSEis whether it can be carried sensitivity They used two tests,based on different in milk. There is no evidencethat milk can [ransmit BSE,but estimate physicochemicalprinciples, so they could assessthe results for there is also no evidencethat it cannot. Now, for the first time, random variation generatingfalse-negatives and false-positives. a long-term collaborative study by scientistswithin the UK's Veterinary LaboratoriesAgency and Agricultural Development The researchersscreened 541milk samplesfrom groups of most and Advisory Servicehas given reassurancethat the cows over severalpregnancies that were BSE-free,or had been sensitivebiochemical testscurrently availablecannot detect fed one dose of either I00 or I g of BSEbrain homogenate. cows. the infectious agentwithin milk from infected Statisticalanalysis of results from22 of the samplessuggested One of the reasonswhy it has taken so long to fi.nd this out is that prions might be present. However, eight of thesewere to do with the difficulty of detecting infection. BSEis caused from BSE-freeanimals and due to the random scatter associated by a protein, called a prion, adopting an abnormal shape. with any biochemical assaySome further false-positiveswere The protein is present in a1lcattle and laboratory detection obsewed using the confirmatory test becauseof a cross- techniqueswere initially unable to distinguish the normal and reaction between a reagentin one of the tests and something abnormal prion forms. Prion levels arevery low in most tissues. in milk collected within the first week post-calving. The first testsfor infective material were bioassaysthat involved The end result of this study is that the researchershave injecting samplesinto mice and then obsewing how their not identified BSEprion protein in the cellular fraction of behaviour and brain structures changed.In late 1999 the first milk from cattle incubating BSEusing tests at their limits of of severalrapid, non-animal testsbecame available. These are detection. This concurs with the current understanding of the now used to detect prions but have not been used to test milk. pathogenesisof BSEand gives further support for the idea that 1 VeroniqueLeplat / SciencePhoto Library milk does not transmit prions.

130 microbiologytoday *ug 06 !!

Bacteriasolve a problemfor wasps Colonizationof buriedplastic

Kaltenpoth, Sabev,H.A., Handley, P.S. & Robson,G.D. (2006). Fungal M., Goettler, colonization of soil-bu ried plasticized po lyvi nyl chloride W, Dale,C., (pPVC)and the impactof incorporatedbiocid es. Microbiology Stubblefield, (2006)152,1731-1739. J.W.,Herznei G., It is difficult to imaginemodern Roeser-Mueller, life without plastics.A panicular benefit comesfrom their K. & Strohm, stability and long-lived nature, sometimesenhanced E.(2005). by including biocideswithin the 'Candidatus plastic. One of the most common plasticsconsist of mixtures of pollvinyl chloride with plasticizers, Streptomyces fillers and stabilizers (pPVC).The downsideis that plastic rubbish philanthi',an survivesfor a very long time in landfill sites endosymbiotic or as an eyesorein the environment. Surprisingly, streptomycete there have been very few studies on how pPVC is degradedby micro-organisms. in the antennae However, researchersfrom the University of Philanthus of Manchesterhave now investigatedhow microbescolonize diggerwasps. abandonedplastics, with intriguing results. lnt J SystEvol Microbiol55, The authorsburied sheetsof pPVC incorporating biocides 1403-141L in fields and in a forestin the Vitoshamountains near Sofiain Bulgaria.Every few months insectshave a love-hate relationshipwith microbes. they retrievedsome of the sheets and testedboth rhe physical Researchershave recently discoveredthat the European stateand the microbesliving on them. After 10 months the thread-like beewolf wasp (Philanthustnangulum) uses bacreria to solve strandsof fungi formed a densenetwork all over the sheets, a problem causedby their nesringhabits. The femalesbuild wrth matching furrows showing how secretedenzymes had nestsin soil provisionedwith paralysedhoneybees. Young eatenaway the plastic. The plasticscontaining biocides were less beewolvesspend the winter within cocoonsin the burrow, thickly coated,but when the tensile-strengthcharacteristics emergingthe next summer to start their own nests.However, were measured,all the sampleshad been weakened the dampnessunderground posesa constantrisk that bacteria by the microbes. or fungi will colonizethe young wasps.This dangeris averted The researchersfocused on the fungi and discovered92 becausethe waspsincorporate symbiotic bacteriainto the different typeson rhe plastics,which they considered cocoonsthat are presumedto produce antifungalcompounds. an underestimate.Some were species of Penicilliumor The bacterialive within glandsin the antennaeof female h.choderma,but many were unidentifiable,even using waspsand are secretedinto the nestjust beforeeggJaying. To moiecular tests.When they testedthe ability of individual follow up their discoveryof this very closerelationship, ire fungi to make use of either of two plasticizers,the researchers researcherswanted to know whether it occurredin all wasn discoveredthat growth increasedover the first 4.5 months, specieswithin the genus Philanthusand the bacteria involvld. but then decreased.They speculatedthat initially there was selectionfor strainsable to feed from Zoologistsin Germanyand the USA collaboratedwrth the unfamiliar plasticizer, but that cell debris and by-products then researchersfrom SouthAfrica to obtain femalesfrom 32 allowed orher fungi to grow on the plastic surface. Philanthusand closelyrelated species. However, the bacteria defied all attempts to grow them in the laboratory. The The value of this researchis that it prolrdes a baselinefor the researchershad to rely on the evidenceof microscopy ways in which plasticsare degradedin the environment.The and molecular methods to identify the bacteria.Electron impact of the differentbiocides on rhis processprovides ideas microscopepictures of sliced antennaeshowed rhat specialized for modifying plasticsro be either more or lessbiodegradable, glands within the antennaewere packed righrly with long fine dependingon their intended uses. strands.This filamentousform of growth is characteristicof actinomycetebacteria and agreedwith the resultsof molecular tests.PCR was used to identify the presenceof a singlespecies ) pPVCbefore (top) and after (bottom) soil burial. Ceoff Robson, of acinomycetebacterium, from the genusStreptomyce.s, within University oJ Manchester antennaeof femalesfrom all the Philanthusspecies. Almost complete 165 rRNA genesequences from bacteriain each A Female European beewolf (Philanthus triangulum) secreting symbiotic' CandidatusStreptomyces philanthi' from specialized wasp specieswere recorded.The high degreeof similarity antennal glands and applyingthem to a brood cell within the soil. amongthe sequencessuggested that the bacteriahad been Erhard Strohm, University of Regensburg, Cermany transmittedfrom mother to offspring for many generations, maybeeven from the origin of this relationshipbetween beewolvesand Streptomyces26-67 million yearsago.

microbiologytoday ;r*g il$ 131 Sooietvwi r^r s silverr-nedol for the seoondyeor rur^tnir-r g of tho ttFlseholseo FlowerSf-r ow Cardening enth usiasts at ln the gardenthere is an unseenand A handoutgiving more detailed ongoing battlebetween plantsand informationabout plant pathogenswas the 2005RHS Chelsea microbessuch as fungi, bacteria and availablefor interestedvisitors to take viruses.Disease-causing microbes away. FlowerShow were keen are alwaysin the environment,ready Thisexhibition was in complete to learnhow some to attackgarden plants when the contrastto lastyear's display where opportunityarises. Fine powdery the Societyhad highlightedthe microbescan harnl residues,grey furry coatings,strange beneficialeffects that microbescan growthsand slimyexudates are signs havein the garden- in the form of their gardenplants. The of microbialattack. plant-rootassociations with rhizobial Society'sexhibit,' Plants SCM staffdesigned, put togetherand bacteriaand mycorrhizalfungi. mannedthe displayfor the Lifelong It wasanother huge undertaking by andmicrobes-adeadly Learningsection in the Creat Pavilion. the Societyand manythanks are due The backdropexplained various aspects to the staffand also membersof the duel' , alsoim pressed the of microbialinfections in gardenplants, SCM who helpedto man the stand includinginformation on how microbes from 8am to 8pm eachday. judges,who awarded attackplants, how diseasesspread, how Overthe sixdays of the show, the plantsprotect themselves from it a silvermedal in the 3,000 leafletswere distributedand infectionand how gardenerscan help the standwas viewed by many Lindley Range. in the fight for healthyplants. -lhe thousandsof interestedgardeners, displayalso included a wide range includingmembers of the Royal of shrubs,perennials, annuals, herbs Family! and fruit bushesset out in attractive JanetHurst containersin a courtyardscene, FayeStokes A TheSCM stand at the 2006 RHSChelsea which were all labelledwith their FlowerShow with MicrobiologyToday SCM EditorCavin Thomas on duty.lan Atherton, susceptibilityto diseasescaused by SCAA bacteria,viruses and fungi.

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producers,comissioning editors, Thefinal was held on 1OthJune at journalists,science communicators and the CheltenhamScience Festival, For-nelobmediatrainers. During the class,each where eachfinalist gave a 5 minute FameLabis a nationalcompetition finalistrecorded a99 secondpodcast presentationof a contemporary to findthe Ul('sbest new talentin on one of the followingthree subjects: sciencetooic that was differentfrom sciencecommunication and this Quantummechanics thosegiven during the heats.Unlike year'scompetition fi nalists included What makesoctopuses different to previousrounds, where only the judges two microbiologists- a virologist other sea life apartfrom their legs? selectedthe best,during the public and a mycologist. As water is made of hydrogenand final,the audiencehad to be - impressed,as they were involvedin Nationalheats were heldduring oxygen why doesn't it burn? choosingthe winner,Jonathan Wood, March and April at Newcastle, l(arlByrne is currentlyin the final a biologistturned Deputy Editor of Swansea,Edinburgh, London and yearof his PhDat Queen'sUniversity Mateilab Today.The winner of the Belfast.Each entrant had only 3 Belfast,where his projectinvolves 2006 competetionreceived L2,OOO, minutesto impressthe judgeswith an lookingfor noveltherapies for viral and will alsoget the chanceto pitch an interestingand excitingpresentation exacerbationsof Chron ic Obstructive ideato Channel4. that was both scientificallyaccurate PulmonaryDisease. He would liketo and engagingto a non-scientific makescience more accessible to non- I FayeStokes audience. Successful contestants went scientistsand enteredthe competiton I PublicAffairs Administrator throughto the afternoonround to aftersome persuationfrom a friend. FameLab(www.famelab.org) is an - oresenta different3 minutetalk this A mycologiststudying for a PhD in initiative of CheltenhamScience Festival time with a publicaudience present! the fungal affectsof rotting wood in partnershipwith NESTAand is Two finalistswere selectedfrom each at the Universityof Newcastleupon supportedby Pfzer, the Daily Telegraph, heatand thesewent on to complete Tyne,Steve Robertson was always Channel4, ResearchCouncils Ul(, the a weekendmasterclass in science scientificallyinclined and wantsmore British Council,Enable lnteractive and 'l9. communication- workingwith TV scienceprogrammes on TV and radio. Silicon Miorobiologyond the r-nedio At a recentmeeting Hugh Pennington looked at how When scientistsare askedto commenton a storythat has microbiologystories get into the mediaand gavesome appearedin the press,Hugh believesthat it is alwaysworth tipson how microbiologistscould publicize their own respondingso thatyou are: research. not accusedof a cover-upat a later date The mediaare interestedin storiesthat havehuman protectedfrom unfair commentsfrom journalists interest.When it comesto microbiology,journalists favour It is importantthat the comment is accurate,professional, lurid diseasesthat couldaffect anybody, especially the not overlyoptimistic and madewithout delay.lt is essentia young or old. Theyparticularly like it if they havea disease that the scientiststays away from sillymedia stunts such that hascaused the deathof a namedindividual. ln 1994 asthe governmentminister feeding a beef burgerto his a televisioncompany ran a storyand issueda pressrelease daughterduring the BSEcrisis. fasciitis'the bug that eatsyou alive' after on necrotizing Whentrying to promoteyour science to the pressHugh there had beena clusterof casesin Cloucestershire.This Penningtonstressed the importanceof gettingacross your was picl

microbiologytoday a.ug {:}6 lf youwould like your name to beadded to our databaseof doesnot depend upon live cell dynamics. Energy transfer can be used for in situ bookreviewers, please complete the bookreviewer interests analysisof molecular associationand can exploit the immensevariety of formon the SCMwebsite. A classifiedcompendium of reviews fluorescencelabelling techniqueswhen from1996 to the presentis also available on thewebsite. applied to permeablizedtissues. The rest of the manual is conventional in its content and approach.The introductory chapter on optics covers the generalbiological microscopist. the theory that is needed to analysethe Unfortunately,segregation of this problems and solutions raised in the material away from the topic of live rest of the manuai. Unfortunately, this cell researchhas resulted in a step is a very big task for one chapter and back from the cutting edgeand a the information may be too condensed more prosaicscope and presentation. to be accessiblewithout considerable However, there are important advances background. The wealth of specific in the microscopy of fixed materials, reagentsthat permit localization of which often are the best approach for different moleculesand structuresin cells many researchquestions. The chapter are coveredin later chapters,including on three-dimensionalmicroscopy non-immunological, immunological (Confocal microscopy,deconvolution, and nucleic acid hybridization-based and structured illumination methods) techniques.Several chapters cover byJohn Murray bucks the generaltrend. protocols specific to eukaryotic model It conveysthe delight and frustration of systems,including yeast,Drosophila, optical analysisof cell structure beyond Caenorhabdtnselegans and cell culture, [he cover siip/sampleinterface. His but not bacteria,protists or common exposition of the interaction of resolution parasites.The final chaptersventure into and contrast over the axial dimension electron microscopy BasicMethods in provides a valuable starting place for In summary this book is a useful Microscopy those dealing with microbiology in the real world. The theory and application referencefor a teaching laboratory or a ByD.L. Spector & R.D.Coldman of a broad range of 3D technologies microscopy facility with very general Publishedby ColdSpring Harbor are compared in a way that can help interestsin a variety of microscopy LaboratoryPress Europe (2005) us make practical decisionsabout our techniquesprimarily for fixed tissues. ' f.45.00 pp.375 researchstrategies. The'Tips'for reliable But, to some extent, it is a useful lsBN0-87969-751-2 imaging and the'Tioubleshooting guide' appendix to its more exciting companion are full of golden advice for users of volume on live cell microscoplz The Cold Spring Harbor Laboratory scanningconfocal microscopes. Manuals grow from the best ground, the Roger Phillips, Universityof Sussex fertile interaction between top scientists Multiphoton microscopy and multiplex- and promising studentsin the intense spectralanalysis are also technologies Celllmaging Techniques laboratory coursesetting. Other manuals on the advancingedge of cell biology on imaging and microscopy from this with exciting applications.However, it is Methods and Protocols series(Live CeIlImaglng: A Laboratory difficult to seea unifying concept in the Editedby D.l. Tattjes& BJ. Mossman Manual and Imaglngin N zuroscience and optical technology or in the biological Publishedby HumanaPress (2005) Dn elopment:A Labor atory Manual) affirm application that would allow these US$125.00pp.512 the excitementof cellular researcharising subjectsto be coveredin one chapter. tsBN 1-58829-157-X from the convergenceof molecular The result is a narrow treatment of and optical advances,while providing eachtechnologr confined largely to a This 2l-chapter volume is tools and cautionary talessufficient for particular microscopeplatform. It lacks complementary Lomost other literature practical results. the practical and theoreticaldetail that availableon the market as it provides might help new usersdo this kind of detailed descriptions of and protocols The BasicMethods inMicroscopy manual microscopy A chapter on fluorescence for a plethora of microscopy and provides good introductions to optical resonanceenergy transfer (FRET)was related methods to study dssues,cells methods and cell labelling techniques notable in its absenceas this is one and macromolecules.By doing so, and many protocols for referenceby (i) powerful sub-micron technology rhat the volume covers different types

134 microbiologytoday aug S6 tr I

of light, (ii) scanningprobe and (iii) recovery deconvolution and tracking basic understanding of the operation singleparticle elec[ron microscopy movement. If all theseterms are alien of an electronmicroscope and do not techniques,including second-harmonic then this is probably not the book for seekhands-on information and./orare imaging, lasercapture microdis-section, you. It is a seriesof robust and well beginnerswith a biological background. fluorescencein situhybridization, laser written descnptionsof the basesof the It is suitable for institutional or oersonal cytometry calcium imaging and near- methods (yes,including equations) purchase. fie1dscanning optical microscopy In and the strengthsand weaknessesof AndreasHolzenburg, Texas A&M terms of the systemsunder study, most each.Physics tends to stay true, so this University of the chaptersfocus on animal tissuesin volume wrll be a longer-term reference a p athology / medical setting. Particularly for anyonewho wants to understand useful,but rare,are the chapterson how thesemethods actually work. In this Reviewson the web evaluating the performanceof laser respectit is excellent. confocal microscopesand microarray Reviewsof the followrng books are lohn Armstrong, Universityof Sussex laserscanners. Regarding the former, the availableon the websiteaL www.sgm. chapterby RobertM. Zucker discusses ac.uk/pubs/micro_today/reviews. cfm on almost60 pages(!) the various PhysicalPrinciples of Opticallmaging and Microscopy quality assessmentaspects in an easily Electron Microscopy Mycoplasmas:Molecu lar Biology, accessiblemanner and provides practical Pathogenicityand Strategiesfor Control By R.F.Egerton instructions on how to test individual Microbe parameters.This comprehensiveand well Publishedby Springer-VerlagCmbH & Bergey'sManual of Systematic rounded book is suitablefor institutional Co. l(G(2005) BacteriologyVolume Two: Part A, B & or personal purchase,novices and f.42.50/US$59.95pp. 122 C2nd edn experts,and it is certainly a must-read rSBN0-38725-800-0 Di ctio n ary of Parasito Iogy for all seriousconfocal microscopistsand This book comprisesa concise imaging facility managers.Unfortunately Molecular MicrobialEcology introduction to the fundamental physical the 19 colour platesare not embeddedin Microbiologyof FreshProduce conceptsof electronmicroscopy and the text, but constitute a l2-page insert LandmarkPapers in Yeast related analyticaltechniques from Biology betweenchapters 13 and 14. Other than a predominantly matenal sciences/ TheBiomedical Scienti

microbiologytoday rrug *6 135 tvorer Thorr-ros Porker {il,1 i Lllffi Wru ffi- P*} ff ffiffiffiffi} M. T.(Tom) Parker, one of the inspirationalfigures of post-war medicalmicrobiology in addressingour increasingconcerns about staphylococcaland other healthcare-associated infections, died on 25 February2005, aged 94. Hiswork on Staphylococcusaureus - initially the type 80/81epidemic strain that caused severe hospital infections in the 1950sand later the increasingantibiotic resistance amongst hospitalisolates leading to the MRSAproblems of the 21stcentury - isas relevant to medicalpractice today as when he ledthe Public Health Labo ratory Service (P H LS) Cross- |nfection Reference Laboratory throughthe 195Osand 1970s.

Tom was born on 27 October 19 12 in North Walsham, Norfolk, establishing services in a ransacked infirmary that had even and obtained his senior schooling al PastonGrammar School lost its water taps. (1923-1931). He went on to Downing College, Cambridge, Early in 1946, on his return to civilian life, he joined the as an exhibitioner, reading Natural Scienceswith specialism newly establishedPHLS under the directorship of G. S. (later in Pathology (I931-1934), gaining fi,rst-classgrades in both Sir Graham) Wilson. Toms first PHLS job was as Director parts of the Natural SciencesTiipos. He then completed his of the Area Public Health Laboratory at Carmarthen, but in medicai studiesat Charing CrossHospital, London, qualifyrng 1948 he moved to Manchester as Consultant Microbiologist MB, BChir (Cantab.) tnI937 ,and continuing there asa House and Director of the Regional Public Health Laboratory. There, Physician. In 1938, he began a Studentship in Pathology at his knowledge and experience were quickly acknowledged Charing Cross Hospital Medical School and gained the Dip- and he was recruited to deliver classesin the ManchesterUni- loma of Bacteriology(London) with distrpction in 1939 from versity Dip. Bact. course as a specialist teacher.His research the London School of Hygiene and Tiopical Medicine under at the time started to focus on S. aureu.sas a cause of severe the exacting standardsof W \M C. Topley and G. S. Wilson. hospital infections and in 1956 his thesis on S. aureu.searned The Second World War interrupted his academic studies. him a Cambridge Doctorate of Medicine. He held strong anti-fascist conviclions and he enlisted in the In 196I, he was appointed Director of the Cross-Infection armed forces at the outbreak of war in 1939. Through to ReferenceLaboratory at the Central Public Health Laboratory, L945, he served in the Royal Army Medical Corps as a Colindale, London, a pos[ he held with distinction until specialist pathologist, initially in the UK and then in India and his retirement in 1978. Dr Parker was pivotal in his role as Burma with the rank of Major. He made lasting friendships Director in bringing together the staphylococcusand strepto- in India with memorable colleagues including James Rhind, coccus reference laboratories working on the qualltative Jerry Morris and Reg Passmore.He had graphic stories of differences in the pathogenic potential of these organisms. his experiences in Lucknow and Calcutta and in his travels Throughout the 57-year history of the PHLS, this laboratory when on leave, with sad accounts of the terrible famine and played a crucial role in investigating and understanding the diseasesthat he wrtnessed at first hand. In Assam, his healthcare-associatedinfections, particularly those caused hospital unit supported Field-Marshai Slim'sadvance and he by S. aureu.sand B-haemolytic streptococci, and helping to was especially proud of his laboratory's record in ensuring devise preventive strategiesthat remain at the heart of the cur- prompt diagnosis and effective treatment of the many rent Department of Health programme Locombat MRSA. Tom patients who developed the debilitating infections that went had directed the laboratory for over half of its first 30 years. with military operations in difficult conditions in a tropical During that time many collaborations were established with climate. His unit was then shipped to Rangoon, arriving on colleaguesand centres overseaswhich formed the foundation the day after the Japanese retreat, and he was faced with of the international links that remain to this day.

136 microbiologytoday ;aug *S Toms unassuming personality, integ- responsibilities.In addition to aspectsof important teaching commitment and rity, wide ra:nge of expertise, and his hospital cross-infection that constantly many authors benefited from'distance friendly and helpful manner endeared required attention, there were innum- learning tutorials' over the re-working him to all his colleagues and his many erable urgent requests to identify the of their papers - ahard act to follow. friends. It was inevitable that his help agents of infectious diseases and to Tom and his wife Beryl were married and influence would be widely sought check their antibiotic sensitivities or in October 1938. Their son David was within the discipline of medical micro- otherwise to guide treatment options. born in 1940 and their daughterJudirh biology, particularly with reference to Tom carried all of these responsi- in 1950. Toms wider interests were staphylococci,strep[ococci and aspects bilities with exemplary calmness and reading, gardening, classical music and of bacterial cross-infection,and he gave real ability opera. He was, however, a dedicated his time unstintingly to students, train- One of Toms greatest legacies to family man and always found time, ees and senior colieagues alike. He medical microbiology was his remark- between all of the other activities for served on a host of internationai com- able service as an editor of, and a hiil-walking with the family, for beach mittees, and his contributions to WHO significant contributor to, our journals days with sandcastles and fossil-hunt- Working Groups, and his Short-Term and to Topiey and Wilson's authoritative ing, and for picking blackberries or WHO Consultantships in the Sudan, text on the Pinciples of Bactenology visiting stately homes. India and Burma (I974, L976,1980), and Immunity, which latterly became After moving to Beckenhamin 1987, all testify to the high regard in which Micr obiology and Micr obial Inf e ctions. He he started on yet another garden and he was held abroad. He had numerous worked tirelessly and with daunting took up serious studies of italian and collaborators across the world. His commitment in these roles. For the Philosophy at Birkbeck College, only daughter Judith recalls a stream of visi- Brh edition of rhe rextbook in 1990, withdrawing from Philosophy classes tors to the family home at Radlett in for which he and Leslie Collier were when, in his late eighties, he felt that his the 1960s, including Lewis Wanna- the two General Editors, he personally essayswere no longer up to s|andard. maker (Minnesota), Theo Poon-King checked each of more than 2,600 pages Beryl died in 1996 and, latterly, Tom (Trinidad) and Hugh Dillon (Birming- of text produced by L25 authors from moved to Sunrise at Frognal House, ham, AL, USA), but there were many, around the world. He worked on five Sidcup, where he made new friendships 'professional many more. He was a Founder Fellow editions of this book (our and found an alpine garden needing of the Royal College of Pathologists bible' over the years) from 1964 to his attention. He had an extended il1- (FRCPath 1964), was appointed a 1998. Those of us who knew Tom's ness with surgery in December 2004, CorrespondingMember of the Deutsche standards in reiation to his editorial but he returned to independent living Gesellschaft fur Hygiene und Mikro- work, with the Journal of Patholog and in Sunrise with his beloved books, his bioiogie (1978), and was President of Bactenologt and subsequently (for nearly paintings, his music and his view of the Hospital Infection Society from 20 years),with the Journal oJ Medical the garden that he had made. He died 1984 to 1988. In 1983, he was award- Microbiolog, found him a demand- peacefully in Queen Mary's Hospital, ed Honorary Membership of the Patho- ing and impressive colleague. We Sidcup. At the funeral, his son, Prof- logical Society of Great Britain and admired him hugely and it was quite essor David Parker, talked warmly of Ireland in particular recognition of his impossible to thank him as he waved his father as a marvellous inspiration contribution to the Societys journals away our expressions of gratitude with and mentor. This is abundantly true for for almost 30 years. In turn, Tom's care a rare smile and a slight flourish of his everyone who met Tom or who worked for members of his staff is epitomized pipe. He was instrumenhl in setting with such a remarkable man. by his support for Winston Maxted, a the standard for scientific rigour and colleaguein his laboratory at Colindale, accuracy of expression in the journal. J. GeraldCollee who was awarded an Honorary Doctor- He expected those standards of his Edinburgh ate from Leiden in recognition of his colleagues, but was also prepared to work on streptococci. spend endless time guiding would-be BrianL Duerden The day-to-day responsibilities of a editors as well as helping innumerable Deparlmentof Health,London hospital laboratory in relation to public aulhors (especially those for whom health were exemplified by Toms work English was not their native language) AndroullaEfstratiou in Carmarthen and Manchester, and to re-work their submitted articles into HealthProtection Agenry, London are reflected in the requirement from worthy scientific presentations. He the current Chief Medical Officer createdthe rehabilitation part of his role (England) that all microbiolo gy labora- as rejection and rehabilitation editor of tories should fulfil their public health the Journal of Medicallvkcrobiolog as an

microbiologytoday sug *S 137 Professor.Jt?Quoyle Ft?S { i m i 1 rewffiffi--}*: :,;ffiffiffiffi} JohnRodney (Rod) Quayle was born and grew up in Mold, North wales.Following his graduation in Chemistryfrom Universitycollege of NorthWales, Bangor in 1945he did a PhDwith ProfessorE.D. Hughes,FRS in physicalorganic chemistry. His obvious talents were recognizedwith a seniorresearch award from the Departmentof scientificand IndustrialResearch and by ProfessorA.R. (later Lord) Toddwho pickedhim to studythe chemistryof bloodpigments in Cambridgewhere he, unusually, took a secondPhD in 1951.lt washis researchon photosynthesiswith ProfessorMelvin Calvin at Berkeley thatignited his career in microbialC1 metabolism.

It was with Calvin that he published the classicpaper on the of formaldehyde incorporation in yeast and rhe idenrifi- carboxylation of ribulose bisphosphate to phosphoglyceraLe cation that the oxidation of methane to methanol in methano- in celi extracts of Chlorellain 1954. Since rhen he had been trophs proceededvia a monooxygenaseusing dioxygen rather recognized universally as being the godfather of rhe subject than water as the source of oxygen in the methanol. who had tutored and inspired many of us with his knowledge Much of this work was done during his tenure as senior and insight which was far broader than carbon metabolism. lecturer (1963-1965) and rhen Professor (1965-1983) ar This lastedright up to the late l980s when his appointmenr as Sheffield University Rod'spioneering work was recognizedby Vice-Chancellor of the University of Bath in 1983 somewhar his election as a Feilow of the Royal Socieryand the award of curtailed his active involvement in the subject, although he the CIBA Medal and Prize of the Biochemical Society,both in did Chair the British National Committee for Microbiology 1978. He servedas Presidentof rhe SGM from 1990 to 1993 (1985-1990). Nevertheless,ir did not diminish his inreresr and was awarded honorary doctorates from the Universities and role as being adviser,confidante and unraveller of some of Gottingen (1989), Bath (L992) and Sheffield (1992). of the more complex issuesof Cl metabolism. Indeed he was But most of all Rod will be remembered by mosr who knew in demand as a plenary speakerlong after his move to Bath. him as the voice of reason,a serious intellect, generousin his Rod returned from Calvin's iab in 1955 with abrie{ foray advice and heip in bringing a compassionareand sympatheric into pyrethrum insecticides at the Tiopical Products Insritute understanding of anyone'sproblems, be they be personal or in London moving swiftly to Sir Hans Krebs' laboratory in scientific. His valedictory lecture ar the I995 spnposium on 1956 to continue his passionfor the metabolism of Cl and C2 microbial growth on Cl compounds in SanDiego was tlpical compounds when he collaboratedwith Hans (now Professor of the man, in which he highlighred all the achievemenrs since Sir) Kornberg and showed that bacterial growth on acerare the first symposium 22years earlier in Edinburgh and played involved the glyoxylate cycle. He used his experience in phoro- scan[ attention to his own discoveries,even though thesehad slmthesis from Calvin's lab with labelled compounds to set out influenced nearly every facet of Cl metabolism for over 30 evaluating the metabolism of methanol, formate and carbon years. In retirement he kept a strong interest in local area dioxide in bacteria.His work led to the discovery of the serine health authority ma[ters and was a member of the Board of pathway and, from studies with methane-oxidizing bacteria, the Bath FestivalsTiust, the Bristol Exploratory andmember the ribulose monophosphate cycle thar paved the way for the of the Council of the Bath Insriture of Medical Engineering. discovery of a variety of cycles and pathways in Cl-utilizing He is survived by his wife Yvonne and children (Susan and bacteriaand yeasts.He alsohad a significant role to play in the Rupert). elucidation of a cyclic pathway of formaldehyde oxidation (the prevailing routes were iinear), the dihydroxyaceton e pathway i Howard Dafton, DEFRA

microbiology todayaug *6 ffiw :: TheWellcome Trust has joined

otherbodies in imposingopen

accessconditions on authors

ofjournal papers reporting

researchthey havefunded.

SCMhas had to developa

strategyto combatthis threat

to theSociety's publishing

income,as Ron Fraser and

RobinDunford explain.

businessmodel, and further elaboration of the author-side payment model may be necessaryThe objective will be to maintain the flow of high-quality submissions, while helping authors comply with their funding agencies' requirements, but maintaining the financial stabihty of the journals. This is important for Society members and for the scienceof microbiology, as it is largely the income from joumal publishing that enablesSGM to support its scientific meetings, numerous granl schemes,and educational and outreach activities. Ron Fraser ExecutiveSecretary

RobinDunford JournalsManager

microbiolo gy today;ru1.; il{i