B I O G R A P H YBrian J. Ford has pub-lished many books andpapers on the develop-ment of the microscope,and in 1981, working inLondon with Sir AndrewHuxley, he discoveredLeeuwenhoek’s original specimens amongpapers in the strong-room at the Royal Soci-ety. He has recreated many of the experi-ments carried out by pioneering micro-scopists, including Leeuwenhoek, Hookeand Trembley. Professor Ford has connec-tions with the Universities of Cambridge,Cardiff, Leicester and Kent and travelsextensively to lecture. He is also well knownas a broadcaster on radio and television, andwas recently awarded a NESTA Fellowship.

A B S T R A C TAntony van Leeuwenhoek (1632-1723) wasa draper and town official in the Dutch cityof Delft. In a career lasting fifty years, helaid the foundation of biologicalmicroscopy. His observation of cells, nuclei,and even bacteria attracted widespreadattention among his contemporaries, andcontinues to attract scepticism today. In1681 he wrote an unambiguous descriptionof the intestinal protozoan parasite Giardialamblia that was subsequently published bythe Royal Society. However, doubt contin-ues to centre on Leeuwenhoek’s ability toobserve fine structures since he used single-lensed (i.e. simple) microscopes. The authorhas recreated Leeuwenhoek’s originalobservations using present-day single-lensinstruments and has shown that it was pos-sible to have observed Giardia using aLeeuwenhoek microscope. Digital micro-graphs and videomicrography have sub-stantiated that Leeuwenhoek’s descriptionswere based on microscopical observation.

K E Y W O R D Slight microscopy, simple microscope, opticalmicroscopy, history of microscope, history ofbiology, Leeuwenhoek, Giardia, photomi-crography, videomicrography, Royal Society

A U T H O R D E TA I L SProfessor Brian J Ford, Rothay House, May-field Rd, Eastrea, Cambridge PE7 2AY, UKTel: +44 (0) 1733 350 888Email: mail@brianjford.comWeb: www.brianjford.com

Microscopy and Analysis 21(4):5-7(UK), 2007

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I N T R O D U C T I O NCould the 17th century pioneer of microscopyAntony van Leeuwenhoek truly see half ofwhat he claimed? This question has beenrepeatedly raised for three centuries – and itre-emerges whenever the history of biologylooks back at his original discoveries.

A topical example is the protozoan parasiteGiardia, which has cause widespread out-breaks in the western world, and which wasfirst described by Leeuwenhoek in 1681. Hisobservations were set out in a letter to theRoyal Society of London dated 9 November1681 (Figure 1). Leeuwenhoek’s letters werealways written in a cheerful, good-naturedstyle in which his descriptions were couched invernacular terms. For all their informality,there is no mistaking the nature of the organ-ism he described that day: “I have sometimesalso seen tiny creatures moving very prettily;some of them a bit bigger, others a bit less,than a blood-globule but all of one and thesame make. Their bodies were somewhatlonger than broad, and their belly, which wasflattish, furnished with sundry little paws,wherewith they made such a stir in the clearmedium and among the globules, that youmight even fancy you saw a woodlouse run-ning up against a wall; and albeit they made aquick motion with their paws, yet for all thatthey made but slow progress.” This is aninstantly recognisable description of Giardia,the intestinal parasite. Leeuwenhoek hadobserved it in specimens of stool he kept whensuffering from diarrhoea.

O U T B R E A K S O F G I A R D I AGiardia is a remarkable little creature, and haslong been a favourite specimen for micro-scopists. Many of the species are harmless com-mensals (I have found them in water from apond rich in shed beech leaves) but G. lambliaparasitizes the human gut. In earlier centuriesit caused a transient infection during child-hood and the immune response meant that itwas less of a problem thereafter. In the mod-ern world, where we live in hygienic homesthat are largely remote from the farm animalsfrom which these parasites are caught, organ-isms like Giardia and Cryptosporidium are lesslikely to be encountered during childhood.

In consequence, when they cause infectionsin later life – particularly in those who areimmunologically compromised – the effectsmay be far more serious. Even so, it is esti-mated that 10% of western people may har-bour Giardia, and the total for the globalhuman population is 20% or more. In someday-care centres, where intimate contactbetween infants is common and diarrhoea isnot unfamiliar, up to two-thirds of the children

may test positive for Giardia.Outbreaks regularly occur in the western

world. There was a small outbreak in Ceredi-gion, Wales, in December 2005, centred on aschool in which transmission was probablyhand-to-mouth. There were eight cases, threeof them symptomless, all among schoolchild-ren. A large outbreak – involving over 2,500cases – was reported from Bergen, Norway, in2004, in which the organism had spreadthrough tap water. Giardia has also beenspread through fruit and vegetables that werecontaminated during preparation, and oneoutbreak caused over 100 guide dogs todevelop diarrhoea.

M O R P H O L O G YThe organism itself is structurally unusual. It isbinucleate and flagellated and has two phasesin the life-cycle – actively motile trophozoitesand highly resistant cysts that are infective.The flattened, leaf-like free-swimming cell isdiminutive, no more than 20310 µm in size(Figure 3). Cysts are smaller, 1038 µm.

Giardiasis can be diagnosed by the micro-scopical observation of the cysts. This is impor-tant in differentiating between this diseaseand other causes of diarrhoea, like Campy-lobacter or Salmonella. The direct examina-tion is done by examining material that hasbeen suspended in zinc sulphate and subse-quently centrifuged to concentrate the cysts.The supernatant specimens are then stainedwith Lugol’s iodine and the cysts can beobserved under oil immersion. A detectionrate of 70% is obtained through this means.

Antony van Leeuwenhoek’s Microscopeand the Discovery of GiardiaBrian Ford, Cambridge, UK

Figure 1: The front page of the letter of 1681 in which Leeuwenhoek reported hisobservations of Giardia to the Royal Society of London [1].

MICROSCOPY AND ANALYSIS JULY 2007 5

Alternatively, the trophozoites can be char-acterised through fluorescence microscopy.For example, cells can be labelled with a fluo-rescent antibody against acetylated tubulin,and they show up clearly against backgrounddetritus. The use of immunofluorescent kitsgives near 90% accurate results, though theyneed to be used by experienced individuals.

Giardia is seen by some as a ‘missing link’between prokaryotes and eukaryotes [4]. Forall their complexity, the cells lack many of thefeatures of eukaryotic cells. There are no mito-chondria, for instance, neither are there gly-cosomes or peroxisomes. Many accounts statethat such structural components are ubiqui-tous in eukaryotic cells: not in Giardia, theyaren’t. There are idiosyncratic tubular ele-ments that seem to lie between the structureof the endoplasmic reticulum and true Golgi.The genus harbours its own parasites, includ-ing mycoplasmas and a double-stranded RNAvirus known as GLV – Giardia lamblia virus. Butit is the binucleated appearance of the cellthat is so characteristic. The paired nuclei looklike the eyes of a clown, peering out from anoval face, and the genus is unmistakable forthis reason alone [5,6].

L E E U W E N H O E K ’ S O B S E R V A -T I O N S The characteristic binucleate appearance ofGiardia, so well known to modern micro-scopists, was not observed by Leeuwenhoek.The single lens of his simple microscopes had aresolution of one micrometre, perhaps evenslightly better than this; the nuclei would beclose to this limit of observation. In addition,they are of marked clarity to the present-dayobserver because we can resort to stainedspecimens.

Today’s microscopists know that there aretwo nuclei, and thus we expect to observethem. Leeuwenhoek had no such preconcep-tion. He reported observing microscopic glob-ules with many living cells, and – if he had seenthem – he might not have considered themworthy of note. There is no doubt about theaccuracy of his observations.

Giardia lamblia does indeed look somethinglike a tiny woodlouse scurrying up a wall. Forall the frantic movements of his flagella, andthe resultant tumbling movement of the cellbody, we observe – as Leeuwenhoek wrote –“for all that they made but slow progress”. Ittumbles as it swims and jostles against partic-ulate suspended matter as it moves, in muchthe way Leeuwenhoek described.

T H E O R I G I N A L L E T T E R Leeuwenhoek’s account was set out in a letterto the Royal Society dated 4 November 1681. Itwas immediately translated into English andread to the Fellows at a meeting on 9 Novem-ber 1681. The most authoritative examinationof Leeuwenhoek’s work and his letters wascarried out by Clifford Dobell, one of thiscountry’s most gifted and skilled protozoolo-gists, who died in 1949. Dobell was able tomaintain parasitic intestinal protozoa in pureculture in his laboratory as no other parasitol-ogist of his day.

The quality of his drawings of microscopicalphenomena was unmatched. Many of thestrange, amitotic forms of cell division that hedelineated were believed to be errors ofobservation or artifacts, and it is only in recentyears that the work of colleagues such as LynnMargulis has confirmed that Dobell’s observa-tions were, after all, accurate [2].

Dobell published his great biography ofLeeuwenhoek in 1932, to mark the tercente-nary of his birth in Delft, and he stoutlydefended Leeuwenhoek. Monica Dobell, whowas much younger than her husband and sur-vived him for many years, took pleasure inreminiscing to me about his life and work, andretelling tales of scientists like Pasteur andFleming who were known to her stepfatherW. S. Bullough. She told me how CliffordDobell ejected that great writer and scientistPaul de Kruif from their London home in WestHeath Road, Hampstead, because he believedthat de Kruif had ‘vulgarised’ Leeuwenhoekby writing a popular account of his life [3].

The late Lord Perry of Walton (Walter Perry)was a good personal friend of mine whoworked alongside Dobell in the 1940s. He toldme of Dobell’s precise and austere manner, hisbrittleness, and his indifference to playing thepolicy game with management.

L E E U W E N H O E K ’ S A C C O U N TAs Dobell made clear, the letter was clearlyoriginal, and the account was unmistakablyaccurate for anyone who has observed Giardiain the living state. And this is how micro-organisms should be observed – not as driedand stained specimens submerged in moun-

tant, but as living entities exploring and inter-acting with their private worlds.

Our modern emphasis on molecular biologyand genetics has an important drawback –biologists have lost sight of how whole cellsbehave, in the round; and this was somethingthat Leeuwenhoek found entrancing. It wasthis very enthusiasm that allowed him toestablish the foundations of microscopy.

Leeuwenhoek used single-lensed instru-ments – i.e. simple, as opposed to compound,microscopes. He typically constructed themout of thin, rectangular metal plates about thesize of a rectangular commemorative postagestamp. The plates he fashioned from brass orsilver which he made for himself. Two plateswere rivetted together, and a perforation wasset into each one, between which he fixed hisdiminutive lens (Figure 2).

Contrary to many current accounts, he didnot use a spherical bead lens made by meltinga drop of glass in the flame. His lenses wereground with jeweller’s polishing paste andwere either planoconvex or biconvex. Some hemade by blowing a glass tube until heobtained a large balloon, the walls of whichwere so thin that it broke into flakes whentouched. At the apex of his balloon forms apellet of glass, and this he occasionally used toprovide an aspheric magnifier with a remark-ably flat field of view.

The best of the nine surviving Leeuwenhoekmicroscopes, now in the collections of the Uni-versity Museum at Utrecht, has one of theseblown lenses. The technique by which theywere obtained was ingeniously worked out bymy late colleague J. van Zuylen of Zeist, The

Figure 3: Stained and fixed Giardia tropho-zoite imaged through oil-immer-sion optical microscope. Colour-corrected public domain micro-graph from the National Institute ofHealth, Japan. Field width 45 µm.

Figure 2: A replica brass microscope ofthe kind made by Leeuwenhoekin the late seventeenth century.Overall length 65 mm.

MICROSCOPY AND ANALYSIS JULY 20076

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Netherlands. Anyone who has tried to blow asmall bulb at the end of a piece of soda-glasswill know how easy it is to blow too hard, andmake just such a huge bubble of glass. Looknext time, and you’ll see the apical ‘nipple’ ofglass that Leeuwenhoek used as an asphericmagnifier.

Of course, such a simplified system makes itcounter-intuitive to conceive how minutemicro-organisms could have been observed.Many accounts point out that Leeuwenhoek’smicroscope was ‘little more than a magnifyingglass’ and this is basically true – though theseshort-focus lenses are capable of surprisinglyhigh degrees of resolution. With early micro-scopes like these I have been able to observemicro-organisms that confirm many ofLeeuwenhoek’s observations.

The smallest structure I observed was latercalibrated under the scanning electron micro-scope at less than one micrometre in width,and thus we know that organisms like Giardiawere theoretically within its compass. It was toexplain these matters that I gave the openingaddress at the First International Conferenceon Giardia and Cryptosporidium conference inAmsterdam in 2004. This was the first meetingto pool expertise on these two important par-asites, each having had a conference of itsown until this date.

During the presentation I demonstratedhow Leeuwenhoek made and used his micro-scopes, and looked back at his first writtendescriptions of Giardia lamblia. It was also pos-sible to present the diminutive micro-organ-isms that can be observed through a single-lensed microscope. The audience were sup-portive and enthusiastic, and everyone foundit revealing to look back at the time whenLeeuwenhoek’s expertise was laying the foun-dations of microscopical biology. Truth to tell,Leeuwenhoek’s work stood by itself and hisremarkable endeavours fitted perfectly withthe tone of the other papers.

H O W D I D L E E U W E N H O E KO B S E R V E G I A R D I A ?There was one question that remained unan-swered. Exactly what had Giardia tropho-zoites themselves looked like through aLeeuwenhoek lens? Nobody knew.

Paradoxically, Clifford Dobell, in document-ing so much about Leeuwenhoek’s life andwork, never used a Leeuwenhoek microscopeeven though he took them apart and drewdiagrams of their construction. And I had notobserved this particular genus, even though Ihad made studies of other organisms of com-parable size. In my presentation I had shownspecimens of the multinucleated frog parasiteOpalina observed through Leeuwenhoek-type microscopes, but this protozoan is largerthan Giardia; indeed it is a relatively giganticcell by comparison.

Professor Huw Smith of Stobhill NHS Trust,Glasgow, one of the meeting’s convenors,offered to provide living trophozoites, so thatI could set out to obtain digital imagesthrough a single-lensed system. I used a tinymicroscope made by the late Horace Dall andcontaining a single lens ground from spinel,

which is characterised by its low chromatic dispersion.

In due course the living Giardia cultureswere received from Professor Smith in Glas-gow, and the investigations began. Theimages were taken with two cameras: anOlympus C-5000 Z camera rated at 5 megapix-els and also a Nikon Coolpix 4500 camerawhich gave 4 megapixels. The organisms werewet-mounted on glass slides under circularcoverslips for direct observation.

In order to restrict the substage cone of illu-mination, Leeuwenhoek used a lamp or a dis-tant window, so in these experiments windowlight was used for some trials, and anunscreened frosted incandescent filamentlamp was used to simulate Leeuwenhoek’slamp. The experiments were successful, andsupported Leeuwenhoek’s remarkable obser-vations. It proved possible to observe livingGiardia trophozoites, and several series of dig-ital micrographs were obtained.

We learn to accept that, when we observemicroscopical phenomena, the brain restoresmany features that may not objectively beclearly evident, just as our sense of hearing caneffectively clean up distorted recordings whenwe listen to music. Once a short-list of imageshad been selected, each was opened in AdobePhotoshop 7.0 and the optical values werenormalised (using the Auto-Levels command)so that brightness and contrast were opti-mised, and seemed closer to the subjectivesense of observation of the living cells.

The results were remarkable (Figures 4 and5). Clearly, Leeuwenhoek could have observedliving Giardia. The cells were clearly seen, theirflagella were apparent and even the twonuclei could be seen in selected fields of view.The results were presented in Chicago at Inter-Micro 05, the annual conference organised bythe McCrone Research Institute, and this paperappeared in The Microscope [7].

Subsequently it proved possible to obtainreal-time videomicrographs of the motiletrophozoites using a Panasonic NV-GS 17 digi-tal camcorder.

Thanks to the encouragement of the para-

sitologists in Amsterdam and the initiative ofProfessor Huw Smith – and guided always bythe extraordinary abilities of Antony vanLeeuwenhoek – it has been possible to recre-ate the discovery of Giardia. We know nowhow accurate was his description, written inNovember 1681. And now we can see imagesthat intimate to the modern microbiologisthow Leeuwenhoek himself must have visu-alised Giardia for the first time in history, backin the Netherlands three-and-a-quarter cen-turies ago.

A C K N O W L E D G E M E N T SAmong the many people who assisted andadvised on the Leeuwenhoek research wereProfessor Huw Smith, Sir Andrew Huxley, MrsMonica Dobell, Mr J van Zuylen, ProfessorPeter-Hans Kylstra and Lord Perry of Walton.The President and Council of the Royal Societyare thanked for assisting me in this work andpermitting me to photograph documentsfrom the Society’s archive.

R E F E R E N C E S1. Leeuwenhoek, A. van, 1681, Ontdeckte onsightbaarhede.

Leiden 1684-1686. [The original description is in a letter inDutch dated November 4, 1681. An English translation wasread at a meeting of the Royal Society in London on Nov-ember 9, 1681.]

2. Dobell, C. The discovery of the intestinal protozoa of man.Proc. Royal Society of Medicine, Section of the History ofMedicine, XIII:1-15, 1920.

3. Dobell, C. Antony van Leeuwenhek and his “Little Animals”:224-5, London: John Bale, Sons and Danielsson, 1932.

4. Soltys, B. J., Falah, M., Gupta, R. R. Identification of endo-plasmic reticulum in the primitive eukaryote Giardia lambliausing cryoelectron microscopy and antibody to BiP. Journalof Cell Science 109:1909-1917, 1996.

5. Nasmuth, K. A homage to Giardia. Current Biology 6:1042,1996

6. Upcroft, J., Upcroft, P. My favorite cell: Giardia, BioEssays20:256-263, 1998.

7. Ford, B. J. The Discovery of Giardia. The Microscope53(4):147-153, 2005.

Content ©2007 Brian J. Ford

Layout ©2007 John Wiley & Sons, Ltd

Figure 4: Living, unstained Giardia lamblia trophozoites imaged through a single-lens microscope magnifying 245x. Image optimised with Photoshop 7 toapproximate more closely to the observer’s view. Field width ~90 µm.

Figure 5: Selective enlargement of the simple microscope view of living Giardiaoffers a remarkably clear view of the flagellar structures and binucleatestructure that characterise the genus. Field width ~60 µm.

MICROSCOPY AND ANALYSIS JULY 2007 7