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McIntosh and Filde's anaerobic jar is an instrument used in the production of an anaerobic environment. This method of anaerobiosis as others is used to culture bacteria which die or fail to grow in presence of oxygen (anaerobes).[1][2]
Construction[edit]
The jar, about 20″×12.5″ is made up of a metal. Its parts are as follows:
- The body made up of metal (airtight)
- The lid, also metal can be placed in an airtight fashion
- A screw going through a curved metal strip to secure and hold the lid in place
- A thermometer to measuring the internal temperature
- A pressure gauge to measuring the internal pressure (or a side tube is attached to a manometer)
- Another side tube for evacuation and introduction of gases (to a gas cylinder or a vacuum pump)
- A wire cage hanging from the lid to hold a catalyst that makes hydrogen react to oxygen without the need of any ignition source
Method of use[edit]
Anoxomat uses the widely known McIntosh and Fildes system of evacuation and replacement to create anerobic environments. During the anaerobic evacuation cycle, oxygen within the jar is replaced with hydrogen. After the anaerobic cycle, a mere 0.16% residual oxygen content is left in the jar, which is then removed by a Palladium catalyst. McIntosh and Fildes anaerobic jar: It is the most reliable and widely used anaerobic method. It consists of glass or metal jar with metal lid, which can be clamped airtight with screw. The lid has two tubes, one act as a gas inlet and the other one as outlet. Additionally lid has two terminals, which can be connected to electrical supply.
- First:
- The culture: The culture media are placed inside the jar, stacked up one on the other, and
- Indicator system: Pseudomonas aeruginosa, inoculated on to a nutrient agar plate is kept inside the jar along with the other plates. This bacteria need oxygen to grow (aerobic). A growth free culture plate at the end of the process indicates a successful anaerobiosis. However, P. aeruginosa possesses a denitrification pathway. If nitrate is present in the media, P. aeruginosa may still grow under anaerobic conditions.
- Second:
6/7ths of the air inside is pumped out and replaced with either unmixed Hydrogen or as a 10%CO2+90%H2 mixture. The catalyst (Palladium) acts and the oxygen is used up in forming water with the hydrogen. The manometer registers this as a fall in the internal pressure of the jar.
- Third:
Hydrogen is pumped in to fill up the jar so that the pressure inside equals atmospheric pressure. The jar is now incubated at desired temperature settings.
References[edit]
- ^Textbook of Microbiology by Prof. C P Baveja, ISBN81-7855-266-3
- ^Textbook of Microbiology by Ananthanarayan and Panikar, ISBN81-250-2808-0
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Pioneersin Medical Laboratory Science
Friedrich Loeffler
1852 - 1915
Loeffler, pupil and assistant of the greatRobert Koch, is justly famed for his part in the discovery of the causativeorganism of diphtheria. One of the most able investigators in thegolden age of bacteriological discovery, he also made many other importantcontributions: he was the discoverer of Loefflerella mallei (thecausative organism of glanders) with Schutz; of the organisms responsiblefor swine erysipelas and swine plague; and of Salmonella typhi in mice. He was an early pioneer in the field of virology. Working with PaulFrosch he demonstrated that foot and mouth disease was due to a filterablevirus - the first recognition of a virus as the cause of an animal disease.
Great technician
Loeffler was a great technician and made manyimportant technical advances. Some of the methods and procedureshe evolved are still in daily laboratory use. Every medical laboratoryscientist must be familiar with Loeffler's methylene blue stain, whichis in widespread use in bacteriological laboratories. He was oneof the earliest to appreciate the value of aniline dyes for staining bacteriaand tissues, and studied them extensively. Loeffler was responsiblefor the introduction of one of the first flagella staining methods.
Perhaps it is in the field of culture media,however, that Loeffler's techniques were most important in his own day. His blood serum medium for the cultivation of the diphtheria bacillus,his malachite green selective medium for B typhosus (S typhi), and hismeat-juice peptone-gelatine medium were widely used. Whilst thesemedia have now largely been replaced by others and by new techniques, developedas a result of much greater knowledge, there is little doubt that theyrepresented a real breakthrough at the time of their introduction.
Though Loeffler was a man of vigorous physiqueand great energy - an indefatigable worker in the true Koch tradition - he published relatively little by comparison with many of his contemporaries. His published work was always prepared with great thoroughness, however,every findings being carefully checked, and it has fully stood the testsof time and confirmation by later workers.
Diphtheria
Edwin Klebs had long searched for the causativeorganism of diphtheria. A professor at Zurich in 1883, he certainlysaw the diphtheria bacillus but, although he saw the organism in diphtheriticmembranes, he was unable to grow it by artificial culture or to prove itsrelationship to the disease. At the time Loeffler took up the searchhe was working in Koch's laboratories. He began his studies by makinga complete histological study of material from a number of fatal casesof diphtheria. In this work he used methylene blue, evolving hisfamous alkaline methylene blue staining solution. His work confirmedthe findings of Klebs and Corynebacterium diphtheriae was known for manyyears as the Klebs-Loeffler bacillus (often referred to as 'KLB').
Loeffler realised that he must do what Klebshad failed to do - find some method of isolating and growing the bacillusin pure culture. Only in this way could he hope to reproduce thedisease in animals and prove the organism's true role. Using a peptonegelatine medium his first attempts at growing the bacillus failed completely. He then turned to a medium consisting of glucose broth and blood serumcoagulated by heat. While this was not selective, the diphtheriaorganisms grew as distinct colonies and Loeffler was able to obtain puresubcultures. The bacilli were the injected into guinea pigs and produceda fatal disease which, on post mortem examination, showed characteristicchanges.
One other very significant fact also emergedfrom these animal experiments; not only did Loeffler show the organismto be the cause of the disease, he also found that it remained localisedat the site of injection in the same way that it remained localised inthe diphtheritic membrane in human cases. this led him to attributethe rapidly fatal effects of the disease to the ability of the bacillito liberate a powerful exotoxin - a theory which he failed to prove. Emile Roux, working with Alexandre Yersin at the Pasteur Institute in Paris,later showed that filtered broth cultures of the diphtheria bacillus, containingno organisms, produced death with the characteristic lesions when injectedinto guinea pigs. This was proof of Loeffler's theory of a powerfulpoison produced by the bacilli and carried throughout the body and provedto be the starting point of Behring and Kitasato's discovery of diphtheriaantitoxin. Loeffler continued his investigations into diphtheriaand discovered the existence of pseudo-diphtheria bacilli, sometimes foundin the human throat and nose. In 1888 and Austrian investigator,Van Hoffmann-Wellenhof, published an account of an organism isolated froma healthy human throat, which he was unable to distinguish from the diphtheriabacillus. Further investigation soon proved, however, that 'Hoffman'sBacillus' was quite distinct from the bacillus responsible for diphtheriasimply another member of the Corynebacterium group.
Loeffler's Life
Friedrich August Johannes Loeffler, the sonof a military surgeon, was born at Frankurt-on-the-Oder on 24 June 1852. He studied medicine at Wurzburg and Berlin, taking his MD in 1874. During the Franco-Prussian War of 1870 he served as an hospital assistant. After passing his stated examination in 1875 Loeffler served as a militarysurgeon in Hanover and Potsdam. In 1879 he transferred to the ImperialHealth Department, coming under the influence of Robert Koch with whomhe worked on steam disinfection and other problems related to public health. In 1886 he became Privatdocent in Hygiene at the University of Berlin andtwo years later was appointed to the Chair of Hygiene at the Instituteof Hygiene at Greifswald, of which he later became Rector. In 1913he followed Georg Gaffky as Director of the Institut fur Infectionskrankheiten(Institute of Infectious Diseases) in Berlin. Loeffler was the authorof the famous history of bacteriology, Vorlesungen über die geschichtlicheEntwickelung der Lebre von den Bacterien. This work, the first onthe subject, covered the history of bacteriology from earliest times untilthe 1880s. It was Loeffler's intention to write a second volume coveringlater developments in bacteriology but, unfortunately, pressure of workprevented him from doing so. With the outbreak of the First WorldWar in 1914 Loeffler returned to the Army Medical Corps with the rank ofGeneral, and was largely concerned with matters of hygiene. He wasnow over sixty years of age and the strains of his important office provedtoo great for his health; early in 1915 he was invalided home, where inApril he died. He was buried in Greifswald.
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Alfred MacConkey
1861 - 1931
The name of Dr MacConkey has been familiarto generations of bacteriologists through the widespread use of the well-knownculture media, which bear his name. The valuable media for differentiatingintestinal organisms of the coli-typhoid group, because of their effectivenessand relative simplicity of preparation, were long favoured by bacteriologists,particularly in the British Isles. MacConkey's media were a developmentof the test which came to be known as the Voges-Proskauer reaction whichhad been introduced in 1898 by two associates of Robert Koch in Berlin,O Voges and B Proskauer.
MacConkey's medium
In the early 1900s, while working as AssistantBacteriologist to the Royal Commission on Sewage Disposal, in the Thompson-YatesLaboratories of Liverpool University and under the direction of Sir RupertBoyce, MacConkey evolved his famous media formulae. His object wasto produce a medium, which favoured the growth of Gram negative organismsyet with an inhibiting effect on those which were Gram positive. At this period knowledge of the intestinal bacteria was far from complete,and the technical procedures for their investigation were still very muchin the developmental stage. The use of fermentation reactions hadbeen established and J E Durham had published his method for the demonstrationof fermentation activity. Conradi and Drigalski were evolving theirselective medium containing crystal violet.
MacConkey's first experiments were with theaddition of one to 1000 parts of carbolic acid to the medium. Thiswas not a new idea; various workers had published media formulae in whichminute amounts of an antiseptic substance were included, and there wasevidence that its presence inhibited the growth of Gram positive organismsmore than the Gram negative ones. After a number of trials MacConkeydiscovered that a suitable concentration with C A hill he added the indicator,litmus. Later, glucose was replaced by lactose.
Acting on a suggestion made by A S Grunbaumand E H Hume, MacConkey replaced litmus with neutral red, which stainedthe colonies as well as being an indicator. (Dr Grunbaum was notGerman, as his name might suggest, but an Englishman who during the 1914- 1948 war, found it desirable to change his name to Leyton). Particularlyin the bacteriological investigation of water supplies, use of MacConkey'smedium in the form of a peptone water without the agar base, was much favouredas a preliminary culture procedure.
The Lister Institute
Alfred Theodore MacConkey, born in1861 theson of a minister in West Derby, Liverpool, studied medicine at Cambridgeand Guy's Hospital. After qualification he went into private practicein Beckenham, Kent, but soon decided to specialise in bacteriology. In 1897 he joined the Bacteriology Department at Guy's Hospital. This was followed by his employment as assistant bacteriologist to theRoyal Commission on Sewage Disposal in Liverpool. When, in 1904,Charles Todd left the staff of the Lister Institute (then the Jenner Instituteof Preventive Medicine) to join the Egyptian Health Service, MacConkeyaccepted an invitation to take his place in London. A year laterhe went to the Institute's branch laboratories at Elstree as bacteriologistin charge.
At the time of MacConkey's transfer the ListerInstitute was going through a period of financial stress and, though theantitoxin preparations produced at Elstree were recognised s among thebest available, the department steadily lost money each year. Onhis appointment to Elstree, the chairman of the governing body (Sir HenryRoscoe) gave MacConkey a clear directive. While the high qualityof the laboratory products must be maintained, the department must notonly be made to pay its way but must also make a substantial contributionto the expenses of the Institute as a whole. This was certainly adifficult assignment but MacConkey, a strong personality, accepted thechallenge with enthusiasm and immediately instituted a policy of stringenteconomy. Research not directly relevant to the production of therapeuticsera was discontinued, staff reductions were made, heating of the laboratorieswas reduced to a bare minimum, and even participation by any member ofthe staff in sporting activities was strongly discouraged as wasteful ofenergy. It is hardly surprising that such measures did little towin him universal popularity, and he acquired the reputation of being 'somethingof a character'. Nevertheless, MacConkey turned the department intoa profitable undertaking, and during the 1914 - 1918 war it had a splendidrecord in satisfying the enormous demands of the armed forces for therapeuticsera.
In 1926 MacConkey retired and went to liveat Brindley Heath, in Surrey, where he died in 1931.
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Patrick Manson
1844 - 1922
The title 'Father of tropical Medicine' hason many occasions been rightly bestowed on this very remarkable Scotsman,who was a pioneer of organised teaching in the undeveloped - but rewarding- field of tropical medicine. He was responsible for the foundationand subsequent development of two famous medical schools, the Hong KongMedical School (which became the University of Hong Kong) and the LondonSchool of Tropical Medicine at the Albert Dock Hospital, now transformedinto the London School of Hygiene and Tropical Medicine and with an internationalreputation. In the 50 or so years of his busy professional life SirPatrick Manson made a series of important discoveries in tropical medicine,but perhaps his greatest contribution was the concept of the role of wingedinsects as the agents responsible for the spread of certain tropical diseases- a concept which paved the way for Sir Ronald Ross's subsequent discoveriesabout malaria.
Filariasis
Although many other investigators - includingJoseph Bancroft in Australia, Wucherer in Brazil, and Lewis in India -played essential parts in the unravelling of the mystery of elephantiasisand its allied conditions, it was Manson in 1877 who showed the mannerof development of the filarial worm Wucheria bancrofti and the role ofthe Culex mosquito in its spread. This was the first occasion onwhich it was proved that an infective disease was spread by animal vectors.
Manson's investigation was greatly helped byhis Chinese servant, Huito. It had been known for some time thatHuito's blood harboured filaria and, very courageously, he volunteeredto sleep in a mosquito cage containing specimens of the mosquito Culexfatigans, the common mosquito of Amoy. It is to be feared that Huitospent a far from peaceful night, but the experiment worked and in the morningManson was able to collect several of the engorged female insects and keepthem in captivity for some days. At intervals he killed specimensand carried out a careful dissection. He quickly discovered thatthe minute worms present in the blood ingested by the insect migrated fromthe mosquito's stomach into the tissue of the thorax, and he noted theremarkable change is size and shape undergone by the parasite. Mansonspent many months performing further experiments to confirm his findings. The research work was carried out under great difficulties: Manson hadalso to perform his ordinary duties, and his problems were increased bythe fact that in China at that time investigation of pathological changesbrought about in the human body by disease was very limited, it being almostimpossible to obtain permission to carry out autopsies. Furthermore,the science of entomology was at an elementary stage of development; fewbooks on the subject existed, and knowledge of the mosquito's anatomy wasalmost non-existent. An appeal to the British Museum for help broughthim a treatise on the anatomy of the cockroach - the best they could do.
Manson's laboratory facilities were limitedto a microscope, a few slides, and simple stains, and he improvised insectdissection instruments using fine pen nibs. Manson also reportedthe nocturnal appearance of the parasite in patients' blood, noting thateven patients with a heavy infestation rarely showed the presence of parasitesin their blood during the day. Some of his critics regarded theseobservations on the nocturnal wanderings of the parasite with such scepticismthat they inquired if the 'Filariae carried watches?' Some years later, when Manson had returned to London, he was able to demonstratethat during the day the parasite embryos collected in the capillaries ofthe lungs.
The lungs fluke
Manson's discovery of human infection by theoriental lung fluke Paragonimus westermani was something of a lucky chance. During a consultation with an important Chinese mandarin on a skin conditionfrom which he was suffering, the patient suddenly expectorated onto thefloor, right at Manson's feet. Disgusted by such objectionable behaviour,Manson was on the point of telling his patient to mind his manners whenhe noticed that the sputum contained a number of rusty brown coloured specks.An immediate examination of the specimen under the microscope revealedthe presence of masses of tiny egg-like bodies. Some time late Mansondiscovered that these bodies hatched out into small embryos. It sohappened that some months previously a Dr Ringer in Formosa had writtento him about the finding at autopsy of a lung fluke in one of Manson'sold patients. Ringer sent the specimen to Manson and further examinationrevealed more of the embryos, which were similar to those Manson had foundin his Chinese patient. This simple observation formed the basisof the study of the parasite which followed in the next few years. When, in 1899, Manson returned to London he continued his studies in tropicalmedicine, including work on trypanosomiasis which brought him into contactwith the tragic figure of Roger Casement, then a member of the ConsularService in Sierra Leone. He was also responsible for descriptionsof several new species of blood filaria, and a demonstration of the lefthistory of the parasite responsible for guinea worm infection. Manson'smosquito-malaria hypothesis was the basis of Ross's subsequent discoveries,and he constantly encouraged and advised Ross in his research. Posterityseems to have been less than just in the recognition it awarded Mansonfor his share in Ross's discovery: without Manson's vision the world mightwell have had to wait for this valuable knowledge.
Doctor, scientist and administrator
Sir Patrick Manson, who filled the roles ofphysician, scientist and administrator was born on 3 October 1844, at CromletHill, Oldmeldrum, Aberdeenshire. He studied medicine at AberdeenUniversity, and soon after qualification joined the Chinese Maritime CustomsService. First he was posted to Formosa and later to Amoy, spendingtwelve years in the service.
In 1883 he moved to Hong Kong where he establisheda large practice, took a leading part in the establishment of medical teachingin the colony, and founded a medical school - now the University of HongKong. When Manson returned to London he opened consulting rooms inQueen Anne Street and, despite the demands of a large practice, continuedhis research. In his house he equipped a small room as a laboratorywhich, from its chronically disorganised state, was referred to by hisfamily as the 'muck room'. With the support of the Colonial Secretary,Joseph Chamberlain, Manson proposed to the Seaman's Hospital Society (ofwhich he was physician) a scheme for the organisation of a School of TropicalMedicine at their Albert Dock Hospital. This important project wassuccessfully launched in 1899, when the first session opened with 27 students. During the three quarters of a century of its existence the school hasgrown both in size and reputation, and is now the London School of Hygieneand Tropical Medicine. Sir Patrick Manson's advice on the problemsof tropical medicine was widely sought and for many years he was consultanton these matters to the Colonial Office. He died on 9 April 1922.
NOTE: This historical note has been abstractedfrom 'Founders of Medical Laboratory Science' and is provided for educationalinterest purposes.
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James McIntosh - 1882 - 1948
and
Paul Fildes - 1882 - 1971
It sometimes happens that the name of an individualis best remembered, and in fact becomes a household word in the laboratory,by a piece of apparatus or a technical method bearing his name, althoughhis achievements in the fields of medical research and discovery may beof much greater importance. The history of the medical laboratorysciences provides many examples of this, including such famous figuresas Pasteur, Koch and Ebrlich. McIntosh and Fildes also fall intothis category. Although both made many important contributions tomedical knowledge they are chiefly remembered for their development ofa new and revolutionary method of achieving anaerobic culture.
Earlier Methods
Since the early days of bacteriology therehad been methods of growing anaerobic bacteria, but none of them was whollysatisfactory as they rarely removed all traces of oxygen. The simplestof these methods was to cover the surface of the culture with a substancewhich was impermeable to air. Alternatively, heat could be used todrive off the oxygen, or a reducing agent such as sodium sulphide - oreven a fragment of living tissue - could be added. Mechanical means(such as a pump) might be used or as in the case of Buchner and Bulloch'smethod, chemical means. Nowadays it is perhaps difficult to appreciatethe relative inefficiency of the methods then available, and how difficultit was to obtain satisfactory cultures of anaerobic organisms. Onlyin exceptional circumstances was the procedure attempted and by no meanscould it be considered a routine measure.
In 1907 Pfuhl published a paper in which hedescribed how he had made use of the catalytic properties of spongy platinumto obtain anaerobic conditions for the culture of organisms. In 1915Laidlaw suggested the use if platinised charcoal and colloidal platinumfor the same purpose. During the First World War the bacteriologyof war wounds, and in particular the investigation of clostridial woundinfection, became a matter of prime importance and it became a matter ofurgency to develop better methods of culturing anaerobic organisms.
Starting from the previous work of Pfuhl andLaidlaw, McIntosh and Fildes developed the highly successful 'McIntoshand Fieldes Jar', which quickly became a standard item of bacteriologylaboratory equipment.
McIntosh and Fildes anaerobic jar
McIntosh and Fildes published their first paperon the anaerobic jar in 1916, entitled A New Apparatus for the Isolationand Cultivation of Anaerobic Micro-organisms. Five years later asecond paper described an improved form, virtually the McIntosh and Fildesjar of today. Though a leading laboratory supply house immediatelymade arrangements to manufacture this apparatus, the paper gave full detailsfor making the jars in the laboratory. A 'do-it-yourself' approachwas prevalent in laboratory practice in those days.
The use of a paint tin measuring seven inchesby five inches, with a lever-off lid, was suggested by the authors. A small stopcock - the type used on model steam engines was found to bevery suitable - was fitted in the lid and carefully made gas-tight withsolder. to the thread of the stopcock was attached a strip of brass,bent to form a bracket. This carried the palladium capsule, whichwas made by impregnation asbestos wool with a solution of palladium chloride,and was enclosed in a small envelope made from wire gauze. Afterimpregnating the capsule was heated in a smokey flame, which coated itwith carbon; finally, complete reduction was brought about by further heatingwith a blowpipe.
After filling the tin with cultures, the methodof use was to fill the gutter of the tin with a thick layer of plasticine,heat the capsule in a bunsen burner, and immediately close the tin andconnect a hydrogen generator to the opened stopcock. In their firstexperiments McIntosh and Fildes had used glass vessels as container, whichmade it possible to judge whether the removal of molecular oxygen was completeby the bleaching of an indicator solution of broth and methylene blue insidethe jar. When tins where used it was impossible to use an indicatorinthe same way; it was found, however, that provided an air-tight seal wasobtained (and it was essential that the stopcock should be well greased)there was little risk of failure.
In 1921 Fildes and McIntosh published a paperdescribing an improved form of anaerobic jar. The main improvementwas the introduction of electrical means of heating the palladium asbestoscapsule. Not only was this a much more convenient arrangement butit had the great advantage that any trace of oxygen which might later diffuseout of the culture medium could be removed easily be reactivating the palladiumcapsule by simply passing current through the heater; if necessary thecurrent could be allowed to run continuously.
McIntosh
Born in Aberdeen in 1882 James McIntosh graduatedMC ChB at Aberdeen University in 1905 and then spent some time at the PasteurInstitute in Paris before returning to Aberdeen in 1908. In the sameyear he moved south to The London Hospital, where he remained until becomingProfessor of Pathology and Director of the Bland-Sutton Institute at theMiddlesex Hospital in 1920. Among many important offices he heldwas a term as President of what was later to become the Institute of MedicalLaboratory Sciences.
McIntosh, alone or in collaboration with other,published well over a hundred papers. His work on syphilis contributeda great deal to the knowledge of the serological diagnosis and treatmentof the disease. He did much work on the Wasserman reaction, whichhad been introduced in 1906. Among his many contributions on thissubject was a paper published in 1912 which gave details of a formula forthe preparation of a cholesterolised alcoholic heart extract, which becamethe standard Wasserman antigen. His work covered a very wide fieldincluding chemotherapy, the Rous sarcoma, tar tumours, dental caries, andviral disease. In his investigation of influenza he became convincedof the role played by a virus in this condition. During the FirstWorld War he turned hiss attention to bacteriological culture media andwas particularly interested in the estimation of pH. McIntosh diedat his birthplace, Aberdeen, in 1948 at the comparatively early age of66.
Fildes
Paul Gordon Fildes, the son of the painterSir Luke Fildes, was born in London in 1882. He studied medicineat The London Hospital, where he subsequently worked as a bacteriologist. In the First World War he served in the Royal Navy and was in charge ofthe pathology laboratory at the Royal Naval Hospital, Haslar. In1934 he went to the Middlesex Hospital as Director of the newly establishedMedical Research Council unit for the study of bacterial chemistry. During the Second World War he was leader of a team of scientists at PortonDown, who were concerned with counter measures against the possible useof bacteriological warfare. After the war the Bacterial ChemistryUnit was reconstituted at the Lister Institute of Preventive Medicine andSir Paul Fildes remained as the Director for three years. He retiredin 1949 and went to the Department of Pathology at Oxford University, workingthere for the next thirteen years.
While Sir Paul Fildes' work on bacterial chemistryearned him an international reputation, he did important work in many otherfields of medicine. His published work included papers on haemophilia,cerebrospinal fever, tetanus, and influenza. His studies on Haemophilusinfluenzae led to the introduction of the special culture medium whichcarries his name. He was recipient of many honours: awarded an OBEin 1919, knighted in 1946, elected a Fellow of the Royal Society and presentedwith the Royal Medal of that Society in 1953. Honorary degrees wereconferred on him by the Universities of Cambridge and Reading. Hedied at the age of 88 in 1971.
NOTE: This historical note has been abstractedfrom 'Founders of Medical Laboratory Science' and is provided for educationalinterest purposes.
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Alexandre Yersin
1863 – 1943
Of all the early pioneer bacteriologist's,the life of the shy, introverted, but adventurous and strong-willed AlexandreYersin was one of the most colourful and exciting. An ardent discipleof the Pasteur School, Yersin's work with Emile Roux in the discovery ofdiphtheria toxin earned him an early reputation as a bacteriologist ofinternational fame. Yersin spent a large part of his working in whatwas then French Indo-China, where he was able to combine the life of aworking bacteriologist with that of an active explorer.
Diphtheria toxin
Although the discovery by Edwin Klebs in 1883of the diphtheria bacillus, and its successful cultivation a year laterby Friedrich Loeffler, were tremendous advances, there were still manyimportant problems of diphtheria left unsolved. At the Pasteur Institute,in the late 1880s, Emile Roux decided to investigate some of these andhe invited Yersin - then a young scientist working at the Institute – tocollaborate with him in this project. The research proved to be particularlyarduous and for three years Roux and Yersin spent long hours in the laboratory,often working far into the night. The results of these labours weremade public in three classic papers published in 1888, 1891 and 1890.
In the first of these publications they confirmedLoeffler's methods of cultivation and made the important observation that,in experimentally infected rabbits, when death did not occur quickly theanimal was often paralysed. Roux and Yersin came to the very significantconclusion that the diphtheria bacillus was capable of producing a poison. They were able to demonstrate this poison, or diphtheria toxin, by passingbouillon cultures of the diphtheria bacillus through Chamberland filters,the sterile filtrate being capable of causing the disease when injectedinto experimental animals.
In their second paper they described in detailtheir procedure of producing the toxin, and reported the results of anelaborate study of its pathogenic action on other types of experimentalanimals.
The technique for the laboratory diagnosisof diphtheria was outlined in their third paper.
The demonstration of diphtheria toxin was toprove of tremendous importance in the treatment of the disease, as it formedthe starting point of von Behring's monumental work on development of animmunising serum.
Yersin's work on plague
In May 1894 plague spread from the mainlandof China to the colony of Hon Kong, where it caused many deaths. Two medical research teams were sent tot investigate and to discover thecause of the epidemic. One of these, the Japanese plague mission,was well staffed and headed by the famous bacteriologist Shibasaburo Kitasato. The other was a small French mission under Yersin. The Japanese teamarrived first and were at work by 14 June, a day before Yersin was ableto arrive in Hong Kong. A laboratory was provided for the Japanesebut Yersin had to be content with a straw hut. Yersin's team consistedonly of himself and tow almost untrained assistants, on of whom promptlydisappeared taking Yersin's modest cash reserves with him. A courtesyvisit to Kitasato was a failure because of language difficulties, and itbecame clear that both missions would have to work independently. Yersin's problems were by no means at an end, however, and everything thatcould go wrong seemed to do so. He had great difficulty in gettingplague autopsy material as it was reserved for the Japanese mission, andit was only after considerable delay that he was able to carry out a fewautopsies.
The rapidity with which Kitasato reported discoveryof the causative organism is well known, yet despite the difficulties heencountered Yersin arrived at the same conclusion within weeks.
The validity of Kitasato's original findingshas been questioned and priority of discovery has been the subject of controversy. rightly or wrongly the Japanese have been credited with the discovery,the work of the unfortunate Yersin merely serving as confirmation of theJapanese findings. One cannot help feeling some sympathy for Yersinwho, with grossly inadequate resources, successfully solved on of the world'sgreatest disease problems just a few weeks too late to claim priority ofdiscovery.
Intrepid explorer
Alexander Emile Jean Yersin was born in 1863in the Swiss town of Aubonne, not far from the shore of Lake Geneva. his father, who had been a professor of natural history, died a few daysbefore his birth. In early life young Yersin showed an interest in,and aptitude for, scientific studies. He began his medical educationat Lausanne then moved to Marburg, and finally completed his training inParis. Yersin had little liking for routine clinical work, and rightfrom the start of his medical career he decided to specialise in pathology. He became an assistant to Andre Cornil, the pathologist at the Hotel-DieuHospital in Paris, and it was while he was here that he suffered an accident,which determined his future medical career.
While carrying out an autopsy on rabies victimYersin cut himself and beacme a potential victim of the disease. fortunately a course of treatment at the Pasteur Clinic prevented the diseasedeveloping, and young Yersin – greatly impressed both by the treatmenthe received, and by the enthusiasm of Pasteur's band of workers – determinedto join them. First, however, he decided to see something of themethods used in Koch's rival school of bacteriology in Berlin, and in 1887spent some months in that famous department. Here he was able tolearn and practice the new technical methods introduced by Koch, and hewas responsible for the introduction to the Paris School of many of themethods evolved in Koch's laboratory. His first research at the PasteurInstitute was concerned with tuberculosis and the action of antisepticand heat treatment on tubercle bacilli – further evidence of Koch's teaching. It was his next three years' work with Roux that were the most important,however:
Once the diphtheria project had been completedYersin's restless nature asserted itself, and he gave up his post and setout to see something of the more distant parts of the world. He obtaineda post as ship's doctor on a French vessel travelling in the Far East,making several voyages to Saigon and becoming fascinated by Indo-China,much of which was still completely unknown. Resigning his post asship's doctor he set off on his first exploratory mission of the country'sinteriot. In the years 1892 to 1894 he made a number if explorations,usually with grossly inadequate resources, but somehow this determinedlittle man survived all the misfortunes which befell him in the courseof his adventurous journeys, and collected valuable information on thetopography, flora and fauna of these regions.
In 1891 Albert Calmette had come to Saigonto organise a branch of the Pasteur Institute and when, after two years,he had to return to France he persuaded Yersin to join the French ColonialMedical Service and left him in charge of the new Institute. Thisposition suited Yersin very well. It gave him an official positionin the country he had come to love and was determined to help. Italso permitted him to pursue both his scientific interests and his loveos exploration. In 1895 he was asked to found a second Pasteur Institutein the country and he chose Nhatrang, a small fishing village in a beautifulbay with a temperate climate and lush vegetaion, where he lived until hisdeath in 1943. In 1933 Yersin was appointed a member of the ScientificCouncil of the Paris Pasteur Institute and once a year made the journeyby air to Paris to fulfil his official duties. The last of his visitswas in 1940, when he was on the last plane to leave Paris for Indo-China.
NOTE: This historical note has been abstractedfrom 'Founders of Medical Laboratory Science' and is provided for educationalinterest purposes.
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Albert Calmette
1863 - 1931
The striking success of modern drugs and methodsof therapy in the control and treatment of tuberculosis has tended to dimmemories of the devastation brought about by that disease less than a quarterof a century ago. The name of the Frenchman; Albert Calmette, isinextricably linked with one of the first successful measures to checkthe spread of this disease. Calmette, with his associate Guerin,produced the BCG vaccine (bacille de Calmette et Guerin) - after nearly20 years' work and this vaccine, with the improvements subsequently madeto it, gave millions of children protection from tuberculosis and savedcountless young lives.
A vaccine to give protection from attacks oftuberculosis was not a new idea; following the successful outcome of vonBehring's work in Germany on diphtheria, and Sir Almroth Wright's workin England on typhoid, it was the ambition of many research workers toproduce equally effective protection against tuberculosis. In 1902von Behring himself had experimented with measures to protect cattle frombovine tuberculosis by the administration of a strain of human tubercleof low virulence. Prior to von Behring's work Others had employed extractsof killed tubercle bacilli, or emulsions of tubercle bacilli cultures whosevirulence they had attempted to diminish by growing the organisms in mediacontaining formalin or similar reagents. The reputation of the Germanbacteriologist, Robert Koch, had been severely damaged by the failure ofhis ill-fated tuberculin as a therapeutic agent.
Pasteur Institute
In 1894 Louis Pasteur, then director of thefamous Pasteur Institute m Paris, was asked by the city of Lille, wherehe had started his career in bacteriology, to establish a branch of thePasteur Institute in that city. Largely on the advice of his deputy, EmileRoux, Pasteur invited Calmette, who was working part-time at the Institutein Paris, to take charge of the new venture. Calmette was well suitedto the post, having previously established a branch of the Pasteur Institutein Saigon, in what was then French Indo-China. Calmette acceptedthe post and in 1895 went to Lille as Director.
While suitable premises were being built hestarted work in temporary laboratories. He was a gifted organiser and withina short time had the laboratories producing sufficient smallpox and rabiesvaccine to meet all the needs of northern France. He also developed a newcommercial process for the conversion of starch into sugar and alcohol,money from which greatly helped in financing the new project. Whenthe work of the laboratories was safely moved into the new building, andvaccine production was in full swing, Calmette turned his attention towhat became his main interest - the discovery of some means of combatingtuberculosis.
BCG
Calmette soon found that the work of the LilleInsatute, and his research into the development of attenuated live vaccinein accordance with Pasteur's practice, required the services of a trainedveterinarian for the maintenance of a stock of experimental animals, mostlybovine. Professor Nocard recommended his assistant, Camette Guerin,for the post and in 1897 Guerin joined Calmette in Lille. They thenbegan the long series of cultures which culminated in the production ofthe successful vaccine, known as BCG.
Over a period of 13 years, using a bovine strainof tubercle which had been isolated by Nocard in 1902 they sub-culturedthe organisms 231 times and the work continued even throughout the Germanoccupation of Lille during the 1914-18 war. By 1908 Calmette andGuerin had succeeded in obtaining an attenuated strain of the bovine tuberclebacillus. The culture medium they used consisted of potato, glycerine,and bile salts. The addition of bile to the medium was to prove of considerableimportance and came about quite accidentally. In the preparation of thefinely divided emulsions of tubercle bacilli needed for the injection ofthe experimental animals, Calmette and Guerin discovered that sterile oxbile was a most effective agent for breaking up the clumps of organisms.Furthermore they discovered that, when bile was included in the culturemedium, changes in the virulence and morphology of the organisms beganto occur. Prolonged culture in media containing bile produced a strainof organisms from which a safe and effective vaccine could be made. The work continued for a number of years, until it was conclusively provedthat the vaccine was harmless to the tuberculosis-susceptible guinea pig. Another ten years were to elapse before Calmette and Guerin were fullysatisfied that it was equally suitable for children. These findingswere duly confirmed by the Academy of Medicine in Paris and the first prophylactictube of BCG on man was carried out in 1921, when a physician persuadedCalmette to allow his vaccine to be used to protect a child delivered ofa tuberculous mother. This unplanned case was completely successfuland Calmette, in collaboration with medical colleagues, then carried outextended - trials. t
In 1924 Calmette and Guerin published theirhistoric paper, in which they showed that trials undertaken in many partsof the world had clearly demonstrated the success and safety of the vaccineas a protective measure. In 1928 a new building was erected at the PasteurInstitute to accommodate the Service du BCG.
The Lubeck disaster
In 1930, just at the time when BCG had gaineda large measure of acceptance and thousands of children had been successfullytreated, a tragic accident occurred. There had always been thosewho doubted the safety of the method, fearing that the strain would notalways remain avirulent, and this disaster immediately brought a strongreaction from its opponents.
The director of the public health laboratoriesat Lubeck, in Germany, had arranged for Calmette to send him a cultureand for a vaccine to be prepared in the laboratories of the city hospital.The batch of vaccine was duly produced and given to some 251 children. Within a year 207 of these children had contracted tuberculosis, and 72died. The whole world was profoundly shocked and Calmette was savagelyattacked both by the medical and by the lay press. Doctors who hadsuccessfully used the vaccine rallied to his side, however, and from Germanythere was particularly strong support. The German health authoritiesheld a painstaking inquiry, which was conducted with scrupulous fairness,and their findings completely absolved Calmette of any blame. Theyfound that the strain of organism supplied by Calmette had been contaminatedin the Lubeck laboratories by a virulent strain, and the German doctorsheld responsible were given prison sentences.
Despite the inquiry's report completely clearingCalmette of any blame, the use of BCG suffered a serious setback for someyears. In Great Britain the safety and value of the vaccine was clearlydemonstrated as a result of a Medical Research Council investigation in1959.
Pioneer of immunisation
Leon Charles Albert Calmette was born in Nicein 1863, the son of a lawyer. His first ambition was to follow a navalcareer, and in due course he went to Brest as a cadet. His studies werecurtailed by an attack of typhoid fever which left him in such a poor stateof health that he was rejected on medical grounds. Calmette did notabandon his ambition to enter the navy and after a period of recuperationwas accepted for training as a naval physician. After qualificationhe saw service in various parts of the world. In 1890 he was transferredto the newly formed French Colonial Medical Service and given leave toundertake further study at the Pasteur Institute in Paris. Here hecame to the notice of the great Pasteur, who was much impressed by hisability and nominated him for the job of organising the Institute in Saigon. Calmette soon had an efficient, if modest, organisation working on theproduction of smallpox and rabies vaccines and he undertook an extensivestudy of snake venoms and the production of anti-venom sera. Unfortunately,after two years he contracted a severe form of dysentery and had to returnto France, leaving the Institute in the hands of Alexandre Yersin (EmileRoux's collaborator in his work on diphtheria) whom Calmette had persuadedto join the French Colonial Medical Service in Saigon.
In Paris Calmette recovered from his attackof dysentery and was given a part-time administrative post at the Ministryof the Colonies. The mornings, and most evenings, he spent workingin Roux's laboratory at the Pasteur Institute and it was while fillingthese posts that the opening in Lille occurred.
During the 1914-18 war, when the German forcesoccupied Lille, all cattle were requisitioned and the bulk of Calmette'sanimal experiments had to cease. He continued, however, to keep somepigeons (in breach of the regulations) and this, and other acts, broughthim into conflict with the occupying authorities. Madame Calmettewas taken to Germany as a hostage, and Calmette himself was interned andonly saved from a possible death sentence by the personal interventionof the highly respected German bacteriologist, Richard Pfeiffer, who atthe time held the rank of Generalarzt in the German army.
On Metchnikoff's death in 1917 Calmette wasnamed, in absentia, Associate Director of the Pasteur Institute in Parisand he took up this post at the end of the war. His death in 1933was undoubtedly hastened by the worry brought about by the Lubeck disaster,and the long enquiry which followed.
His associate in the discovery and developmentof BCG, Cermelle Guerin, was born in Paris in 1872 and trained as a veterinarian. Almost all his professional life was spent in the service of the PasteurInstitute in Lille, and later in Paris, where he continued to supervisethe production of BCG after Calmette's death. Before his death in1961 Guerin had the satisfaction of seeing the final acceptance of BCGas a safe and efficient prophylactic measure against tuberculosis.
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Wilhelm Bunsen
1811-1899
Wilhelm Bunsen, the famous 19th century Germanchemist and physicist, made many important discoveries and contributeda great deal to the advancement of knowledge in his chosen field. He is, however, most widely known by the gas burner which bears his name. This burner, which was invented more than a hundred years ago, is stillan indispensable piece of equipment in many widely different types of laboratory.Initially designed with the needs of the chemist and physicist in mind,it has proved essential in medical laboratories also, and especially inbacteriology. The pioneer bacteriologists were fortunate that theburner was available in the last quarter of the 19th century, when tremendousadvances were made in that science.
While the Bunsen burner has found. universalapplication, and perpetuated the name of its inventor far outside the worldof science, many of Bunsen's other discoveries were also of great importance. He was a prodigious worker and, although many of his researches were long-termprojects, he published over a hundred scientific papers.
An explosive subject
Bunsen spent forty years as Professor of Chemistryin the University of Heidelberg.
It was in the early years of his professorshipthat he invented his famous burner.
Many others before him had tried to devisean effective laboratory burner, using coal gas, but none of these effortshad proved satisfactory; in fact, some had been literally explosive failures.With the exception of the simple spirit lamp and the charcoal fire, theonly available alternative was an oil burner (known as the Argand) whichhad originated in Switzerland in the 18th century. In the 1850 thelaboratories of the
University of Heidelberg were moved from therather ramshackle remains of a long-disused monastery to a new buildingin the Plockstrasse. This move coincided with the introduction ofcoal gas to the city and the university authorities decided that theirnew laboratories should be connected to the gas supply.
It was in the early years of his professorshipthat he invented his famous burner.
Many others before him had tried to devisean effective laboratory burner, using coal gas, but none of these effortshad proved satisfactory; in fact, some had been literally explosive failures.With the exception of the simple spirit lamp and the charcoal fire, theonly available alternative was an oil burner (known as the Argand) whichhad originated in Switzerland in the 18th century. In the 1850 thelaboratories of the
University of Heidelberg were moved from therather ramshackle remains of a long-disused monastery to a new buildingin the Plockstrasse. This move coincided with the introduction ofcoal gas to the city and the university authorities decided that theirnew laboratories should be connected to the gas supply.
Bunsen was well versed in the conditions requiredfor the combustion of a co gas and air mixture and, despite the seriesof failures experienced by other worker who had attempted to constructa practical gas burner, he was fully convinced that such a device was possible. Needing a hot, smokeless flame for some experiments on spectrum analysishe resolved to design a burner which would give the kind of flame he requiredwithout any danger of an explosion. In this enterprise he was assistedby one of his pupils, Henry Roscoe, who later became Professor of Chemistryin Manchester and was knighted. The team also included Peter Desaga,a technician on the university staff.
Painstaking research
Experimental work on the project began in 1853. Superficially the problem appeared a simple one; it was necessary merelyto convert a bright, smoky flame into a hot, smokeless one. Yet itwas only after two years of delicate and painstaking experiment that successwas finally achieved, with the production of the familiar Bunsen burnerThere were two basic difficulties which had to be overcome. First,they had to determine the correct mixture of gas and air (they eventuallysettled on three volumes of air to one of gas): secondly, it was necessaryto ensure complete burning of the gas and to minimise the amount of carbonproduced. This was done by admitting air into the flame from below,so that there was air both inside and outside the flame.
The principle of the Bunsen burner was subsequentlyemployed in the domestic gas stove, and indeed in any gas apparatus requiringthis-kind of flame. Several modifications of Bunsen's original designappeared, mostly intended to give a larger area of hot flame, and a pilotlight was also subsequently included.
Spectrum analysis
Many people consider that Bunsen's most importantfindings arose from the long series of successful investigations in thefield of spectrum analysis which he carried out in conjunction with hiscolleague at Heidelberg, Gustav Kirchoff. Yet these were not hisonly noteworthy discoveries. Before he went to Heidelberg, Bunsenhad been employed at Cassel and as Professor of Chemistry at Marburg. During this phase of his life he invented the carbon-zinc battery (thenow obsolete 'Bunsen cell'). He also conducted extensive researchinto the properties of the cacodyl compounds. In the course of thislatter work an accident occurred which almost cost him his life, and whichresulted in the loss of an eye.
Other areas of research which continued tointerest Bunsen for many years were the development of methods for theaccurate measurement of gaseous volume, and the investigation of gaseousdiffusion and absorption. Unlike many of his contemporaries Bunsenwas no compiler of manuals; the only book he ever published was one describinghis gasometric researches. His years of work with his friend Kirchoff,who also held a chair at Heidelberg, established spectrum analysis as ascience and led to Bunsen's discovery of the rare elements caesium andrubidium. He also observed the high luminosity displayed in the colourless flameof certain rare earth metals, a discovery which was to be of immense valuein the incandescent gas mantle industry.
Liked and respected
Bunsen is said to have been a kindly and modestman, greatly respected by his contemporaries in the world of science andbeloved by the students who came to him from all parts of the world. He spent 37 years as Professor of Chemistry at Heidelberg and during thattime received many offers of more attractive appointments in larger universitycentres. All these invitations he refused, preferring the quiet andrestful atmosphere of Heidelberg.
Born on 31 March 1811 in the city of Gottingen,on the eastern side of the Prussian province of Westphalia, Bunsen wasthe son of the chief librarian and Professor of Classical Philology atthe University of Gottingen. Young Bunsen was educated in his hometown and afterwards furthered his knowledge by periods of study in Berlin,Paris and Vienna. At the age of 22 he returned to Gottingen to rakeup a post in the university. Three years later he moved to Casseland in 1838 he was appointed Professor of Chemistry at Marburg. In1861 he moved to Breslau, and in the following year to his final appointmentat Heidelberg. Bunsen retired from active university life in 1889and remained in Heidelberg until his death ten years later.
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David Bruce
David Bruce
1855-1931
Sir David Bruce was one of the great pioneersof the 'Golden Age of Bacteriology, and although be is best rememberedfor bis outstanding discoveries in connection with two diseases-Malta feverand sleeping sickness-be also carried out important research in other fields. As well as being a great scientist he was a gifted medical administratorwho had a distinguished military career.
Malta fever
Malta, or Mediterranean, fever was renamedundulant fever when its wider geographical distribution became appreciated;the latter title being derived from the character of the patients' temperaturecharts. The disease, probably first introduced into Malta duringthe Crimean war, quickly became a permanent scourge of the island witha high death rate-the result of large numbers of both the civilian andgarrison populations suffering attacks of this lengthy and debilitatingillness. Bruce's discovery in 1887 of the causative organism, andthe part he played later in demonstrating the role of the goat in spreadingthe fever, eventually resulted in the eradication of the disease from Malta.
It was while serving in Malta as an armymedical officer that Bruce became concerned with the problem of Malta fever. He was convinced that the disease was due to some form of bacterial infectionand concentrated his efforts on a search for the causative organism. Within two years he was successful for, in the spleens of patients whohad died of the disease, he discovered a 'micrococcus', and by carefulexperiment proved conclusively the aetiological significance of the organism,which he called Micrococcus melitensis. In 1920 Tensier and Meyerplaced the organism in a separate genus, with the name Brucella in honourof its discoverer, and it became known as Brucella melitensis.
Bruce's discovery of this organism had littleimmediate effect on the control of the disease, but the introduction in1897, by Sir Almroth Wright and Frederick Smith of an agglutination testfor the diagnosis of undulant fever was the first step in bringing it undercontrol. In 1904, twenty years after his initial discovery, Brucereturned to Malta and played an important part in the final conquest ofthe disease. He headed the Royal Society's Malta Fever Commission,which proved that the principal source of infection was contaminated goats'milk.
Sleeping sickness
While several workers contributed to the discoveryof the cause of this dreaded tropical disease, Bruce's work was by farthe most important. His earlier finding that a trypanosome was the causeof a disease in cattle and horses led directly to the discovery that sleepingsickness was also a form of trypanosomiasis. Later he played a leadingrole in the work that demonstrated the part played by the tsetse fly inthe transmission of the disease.
Bruce's interest in trypanosomiasis began in1895 when he was stationed in South Africa. At the special requestof the Governor of Natal, Sir Walter Hely-Hutchinson who had been LieutenantGovernor of Malta at the time of the successful research into the causeof Malta fever - Bruce was sent to investigate a disease of domestic animals,called 'nagana', which caused a high mortality amongst horses and cattle. At first Bruce regarded the problem as bacteriological but, when this lineof investigation proved fruitless, he switched his attention to the examinationof blood films. Unfortunately his laboratory resources were limited. The only stain he had available was carbol-fuchsin - not a particularlysuitable blood stain-and although he did find some curious objects betweenthe blood cells he regarded them as artefacts. The search was, however,much more rewarding when he examined wet preparations of blood and foundan organism which, at first, he thought might be a filaria. Thisorganism was later named Trypanosoma brucei and, by experimental infectionof test animals, Bruce proved it to be the cause not only of 'nagana',but also of another condition of domestic animals-tsetse fly disease; infact, he established that they were the same disease. Recalled tomilitary duties in January 1895 Bruce's research was interrupted, but laterin the year he was able to return and complete his work on trypanosomiasisin domestic and wild animals, and on the part played by the tsetse fly,Glossina morsitans, in transmitting the infecting organism.
In 1902, the War Office seconded Bruce to theRoyal Society's Commission on Sleeping Sickness, in Uganda. Workingfor this body he played a prominent role in proving that the disease wasa form of trypanosomiasis, transmitted by the tsetse fly Glossina palpalis. This established the basic facts about the aetiology and epidemiology ofsleeping sickness and was the foundation upon which all subsequent investigationswere based.
Scientist and soldier
Sir David Bruce was born in Melbourne on 29May 1855. His father, who was an engineer connected with the miningindustry, was only a temporary resident in Australia and when David Brucewas five years old the family returned to Scotland and settled in Stirling.
Bruce entered Edinburgh University in 1876,graduating in medicine in 1881. His first post was as an assistantto a doctor in Reigate but within two years he decided to enter the ArmyMedical Service and was commissioned in 1883.
Almost immediately he was posted to Malta wherehe did his famous work on Malta fever. His next appointment was thatof assistant professor of pathology at the Army Medical College at Netley,where he stayed for the following five years. Before taking up this post,however, he spent his leave in Berlin working in Robert Koch's laboratories.
In 1894 he was posted to South Africa and commencedhis investigations into trypanosomiasis. While in South Africa hewas recalled to purely military duties during the Boer War. Shutup in Ladysmith during the siege, he took charge of a large military hospitaland his wife, who accompanied him on all his travels, undertook the dutiesof sister-in-charge of the operating theatre. Sir David does notappear to have had a great liking for the finer details of laboratory techniqueand Lady Bruce, who became a highly-skilled bacteriologist and microscopist,was largely responsible for the technical work that his research required.
During the 1914-1918 war Bruce was commandantof the Royal Army Medical College at Millbank and was responsible for researchinto trench fever and tetanus. He was knighted in 1908 and receivedmany honours to this day his name is perpetuated by the David Bruce Laboratoriesin Wiltshire, and the David Bruce Hospital in Malta (formerly the militaryhospital). He died in London on 27 November 1931, four days afterthe death of his wife.
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Jules Bordet
1870 - 1961
The decade 1870-1880 is often referred to as'The Golden Years of Bacteriologic Discovery'; one might, with equal justification,label the years 1890-1901 as 'The Golden Years of Immunological Discovery'. Famous figures such as Emil Von Behr his associate Shibasaburo Kitasato,Paul Ehrlich, Elie Metchnikoff, and Richard Pfeiffer are among the manywho founded the new science of immunology.
It was a well-known fact that, following recoveryfrom certain infections, individuals acquired protection from subsequentattacks, and that in some instances this protective power could be stimulatedwithout an attack of the actual disease. The practice of vaccinationagainst smallpox (introduced by Edward Jenner) and Louis Pasteur's workon anthrax and rabies clearly demonstrated the value of this knowledge.Understanding of the underlying mechanism of antigen-antibody reactions,and evaluation of methods of serological diagnosis, were the aims of thepioneers of immunology.
In this new field the Belgian Jules Bordet,together with his fellow countryman Octave Gengou, played a leading role. Their researches were on the properties of serum from immunised animals,and the Bordet-Gengou complement fixation technique was later the basisof a number of diagnostic serological tests. Bordet was not onlya great immunologist, he was also an eminent bacteriologist and, with Gengou,was responsible for the discovery of the causative organism of whooping
cough.
cough.
The complement fixation reaction
Bordet's early interest in the field of immunitywas shown in a paper published in 1895. In this he demonstrated thattwo quite different substances were involved in the phenomenon of bacteriolysis. These substances he called 'alexine' (from the Greek, I ward off) and 'substancesensibisatic'; terms which were replaced by Ehrlich's 'complement' and'amboceptor'.
Bordet utilised the work carried out in thepreceding year by Richard Pfeiffer and Vasiliy Isayeff, who had demonstratedthe possibility of using immune serum as a diagnostic test for cholera.Pfeiffer and Isayeff had found that cholera vibrios injected into the peritonealcavity of an immunised guinea pig quickly lost their motility and wereeventually phagocytosed by leucocytes. This discovery became knownas 'Pfeiffer's phenomenon', and Bordet and Gengou used the same principlefor their in-vitro tests.
In 1901 Bordet and Gengou published detailsof their complement fixation test. This method, based on their previouswork on immune haemolysis and bacteriolysis of cholera vibrios, was applicableto the detection of antibodies to a variety of bacteria, and gave riseto a most important range of diagnostic serological tests. Bordet and Gengou'scomplement fixation method also had applications in the evolution of atechnique for the detection of human blood in medico-legal work.
Causative organism of whooping cough
As a research team Bordet and Gengou provedan ideal combination and in 1906, with the publication of a paper on thecausative organism of whooping cough, they made a further major researchcontribution. In this paper, Le Microbe de la Coquelusbe, they gavea full account of the properties and cultivation of the organism now knownas Bordetella pertussis, together with strong evidence supporting its aetiologicalrelationship to the disease. The organism they isolated was a minutecoccobacillus which they found in sputum expectorated during paroxysmsof coughing in acute whooping cough. For the cultivation of the organismthey devised a special slope culture medium known as Bordet and Gengou'smedium, the principal constituents are potato extract, agar, and blood.
In modern bacteriological nomenclature thegenetic part of the organism's name-Bordetella-is a tribute to one of itsdiscoverers. Although for many years Bord. pertussis was known as Haemophiluspertussis, it was originally called the 'Bordet-Gengou bacillus'
Jules Bordet
Born on 13 June 1870 in the Belgian town ofSoignies, some twenty miles from Brussels, Jules Bordet studied at theUniversity of Brussels and graduated as a doctor of medicine in 1892. Two years later he moved to the Pasteur Institute in Paris, where he heldthe post of 'preparateur' in Metchnikoff s laboratory. In 1901 hewas recalled to Brussels to found the Pasteur Institute and to become itsfirst director. and in 1907
he was appointed professor of bacteriologyin the University of Brussels-a post he held until his retirement in 1935.
he was appointed professor of bacteriologyin the University of Brussels-a post he held until his retirement in 1935.
During his long and distinguished career Bordetreceived many honours, including the Nobel Prize for medicine and physiologyin 1919. Although Bordet and Ehrlich held very different views onthe mechanism of the antigen-antibody reaction, this difference of opinionwas never allowed to degenerate into bitter controversy, as sorry timesarose between distinguished protagonists of opposing theories. JulesBordet outlived most of his contemporaries: he was 90 years old when. m1961, he died in home at Ixelles on the outskirts of Brussels.
NOTE: This historical note has been abstractedout of 'Founders of Medical Laboratory Science' and is provided for educationalinterest purposes.
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Emil von Behring
1854 - 1917
It is often held that the discovery of antitoxicimmunity was one of the greatest achievements of medical science. Certainly the discovery of diphtheria antitoxin, offering dramaticallysuccessful treatment of a disease which caused thousands of deaths amongstyoung children each year, was an epoch-making event.
The nature of immunity had for a long timebeen the subject of deep controversy. There were two main schools of thought.The 'Solidists', led by the great Elie Metchnikoff, thought that phagocytesplayed the leading role. Supporters of the alternative theory, the'Humoralists', considered that a substance evolved in the body was mainlyresponsible.
Von Behring's work gave tremendous supportto the 'Humoralists'. He proved that the serum of an animal whichhad recovered from an attack of a certain disease could be injected intoa second animal and give it immunity to that particular disease Thiswork clearly established that some protective substance was present inthe serum, which would neutralise toxins produced by certain infectingorganisms. This substance Behring called antitoxin, and he was thefirst to use this word.
Many others made important observations thatled to the discovery and development of diphtheria antitoxin- Loeffler,Roux, Yersin, Ehrlich, Kitasato, and probably Robert Koch-but the actualdiscovery was undoubtedly the work of Behring and his Japanese co-worker,Kitasato. What is perhaps equally remarkable is the rapidity withwhich it became a widely used practical measure. In l890 Behringand Kitasato published the result of their research into diphtheria andtetanus antitoxins, showing how they had cured infected animals and immunisedhealthy animals. Exactly one year later, on Christmas night 1891,a child was successfully treated in a Berlin clinic with diphtheria antitoxin.
Diphtheria antitoxin
Behring's search for an antidote to the toxicproducts of bacteria started when he joined the team of workers in RobertKoch's laboratories. As an army surgeon he had had considerable experienceof iodoform, an antiseptic dressing then used routinely in the German army. He had formed the opinion that its beneficial action was not solely dueto any direct antibacterial power, but to other properties it had of neutralisingthe toxic products of septic bacteria. Roux and Yersin, in Paris,had already demonstrated that diphtheria brought about its lethal effectsby the liberation of a toxin. That it was possible to acquire immunityto diphtheria had been shown by Loeffler, who had also worked in Koch'slaboratory.
Another worker in Koch's laboratories was Fracnkel,who at this time was attempting to discover a method of attenuating diphtheriabacilli to produce a vaccine. Behring's first experiments were onthe lines of Fracnkel's work and were, no doubt, influenced by his experienceof iodoform. To cultures of diphtheria he added very small amountsof iodine trichloride, and by this method he did have some success in protectingguinea pigs. His next step was to render guinea pigs immune to diphtheriaby repeated injections of the exudate from an animal dead of the diseaseand containing no diphtheria bacilli, but only the toxins. He showedthat sub-lethal doses of diphtheria toxin had an immunising effect..
At this time Shibasaburo Kitasato, a Japanesebacteriologist, was also working in Koch's laboratory. He had recentlydiscovered the causative organism of tetanus an, had shown that it produceda lethal toxin, and was actively engaged in the search for tetanus antitoxin. Behring and Kitasato joined forces, and in December 1890 published a paperon their work on tetanus and diphtheria antitoxins. Almost concurrentlyBehring published a separate paper, giving details of his work on diphtheriaantitoxin. Within a matter of months Behring succeeded in workingout a method of rendering guinea pigs immune to diphtheria, or of curinginfected animals by the intraperitoneal injection of blood from immunisedanimals, and at the Congress of Hygiene in 1891, in London, he made a publicannouncement of this work.
During the following three years much progresswas made in methods of producing diphtheria antitoxin, and establishingthe correct dosage and standardisation. In these measures Paul Ehrlichhad a great deal of influence, and it was during this period that the horsewas first used for the production of antitoxin. At the InternationalCongress of Hygiene and Demography at Budapest in 1894 Roux fully confirmedall Behring's claims, and diphtheria antitoxin began to be readily availablefor general clinical use.
During the next fifty years many improvementsin the specific treatment of diphtheria by antitoxin took place. In 1912 Behring was one of the first to explore the possibilities of activeimmunisation, and in some countries a policy of active immunisation ofchildren was adopted. The almost complete eradication of diphtheriafrom Britain, however, had to await an official campaign in the 1940s.
Von Behring's life
Emil Von Behring was born in the small townof Hansdorf in East Prussia on 15 March 1854; by a strange coincidence,a day after the birth of his associate in later life, Paul Ehrlich. Behringstudied at the Army Medical School in Berlin and graduated in 1878. The first ten years of his professional life were spent in the German ArmyMedical Service, working at various military establishments, and in 1888he became lecturer at the Army Medical College. In 1889 he was appointedassistant to Robert Koch at the Institute of Hygiene, and within a yearmoved to the Institute of Infectious Diseases as Koch's assistant.
In 1893 the title of professor was conferredon Von Behring in recognition of his work on diphtheria, and two yearslater he moved to Halle and occupied the Chair of Hygiene. This, however,was to be only a temporary post. Within a year he accepted the postof Professor of Hygiene and Director of the Hygiene Institute in the Universityof Marburg, when only 41 years of age.
Commercial manufacture
Behring became interested in the commercialmanufacture of antitoxin, and the German organisation Farbwerke Höchstbuilt and equipped an extensive plant for this purpose.
The early diphtheria antitoxins had many seriousdisadvantages, and some dangers, which research overcame in the next fiftyyears. The commercial production of diphtheria antitoxin was quicklyestablished, however, first in Germany and shortly afterwards in many othercentres. In England production began in London in 1895 in what isnow the Lister Institute.
Another large combine, the Behringwerk, developeda bovo-vaccine which Behring had devised for the immunisation of cattleagainst tuberculosis. Although this vaccine was at the time widelyused in many parts of the world, the results of prolonged experience weredisappointing. As a result of these commercial connections Von Behringbecame a rich man and a large landowner and on his extensive estate hemaintained a big herd of cattle on which his tuberculosis immunisationprocess could be tested under his direct supervision.
In 1901 Von Behring was awarded the Nobel Prizefor Medicine and in Germany his work received official recognition by theconferring of the title 'Excellency' on him. The Paris Academie de Medécineawarded him a prize of £1000 and the Institute of France £2000. Emil Von Behring died, following an attack of pneumonia. on 31 March 1917at the age of 61, and was buried in Marburg.
NOTE: This historical note has been abstractedout of 'Founders of Medical Laboratory Science' and is provided for educationalinterest purposes.
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Ernst Abbe
1840 - 1905
Improvement in the performance of the microscopeproved to be of paramount importance in the emergence of the medical laboratorysciences in the last part of the 19th century. This in turn demandedtremendous progress in methods f or the preparation of specimens for microscopy,and new methods of staining tissues and bacteria. Many famous characterscontributed to the evolution of the microscope from the first crude, single-lensinstruments of the 17th century to the highly practical compound models,with achromatic lenses and much improved mechanisms, of the mid-19th century. It was, however, the German microscopist and mathematician, Ernst Abbe,who did most to lay the foundations for the development of the modern instrument. If he opened up new fields for microscopy by introducing the oil-immersionobjective and the apochromatic lens, and initiated new manufacturing processes,replacing the manual methods of individual craftsmen with mass productionof precision-built instruments.
Oil-immersion objectives
Abbe was not the originator of the oil-immersionobjective, but he was the first to produce a really practical system forits use. In 1873, six years before Abbe published his famous paperOn Stephenson's system of homogeneous immersion of microscope objectives,an American instrument maker called Robert Tolles devised and produceda 1/10 inch immersion objective, using Canada balsam as the immersion medium.Tolles' invention did not meet with a great deal of success as the objectiveitself was a clumsy affair and Canada balsam a messy and inconvenient mediumto work with. Also, as Tolles had an unfortunate manner of expressing himselfin print his views were much misunderstood and this discouraged the furtherdevelopment of a promising project.
The immersion principle was not a new idea. Robert Hooke, microscopist and curator of the Royal Society in the 17thcentury, recorded having used little globules of liquid with his single-lensmicroscope. The idea does not appear to have been developed any further,however, until the Italian mathematician and physicist Giovanni Amica,experimented in the 1850s with various immersion fluids before finallydeciding on water. Later the optician Edmund Hartnack fitted waterimmersion objectives with correction collars, and in the 1860s these lenseswere produced in considerable numbers, particularly in continental Europe.
In 1878 an eminent English microscopist, JW Stephenson, suggested the use of an immersion fluid with similar refractiveand dispersive powers to glass. Unfortunately Stephenson lacked thetechnical knowledge and the workshop facilities required to develop theidea, and he therefore wrote to Abbe suggesting that he might like to explorethe possibilities. Abbe was very impressed by the scheme; he had consideredsuch a project some years previously, but had not investigated the ideafurther. Stimulated by Stephenson's letter, and with the facilities ofthe Zeiss organisation at his disposal, Abbe developed the scheme and withina few years the oil-immersion high-power objective had become a standarditem of equipment on all but the cheapest microscopes. The searchfor a suitable immersion medium presented some difficulty, and over 300fluids were investigated before cedar-wood oil was selected.
Apochromatic lenses
Despite the usefulness of achromatic lenses,Abbe was well aware that it was not possible to correct completely forboth chromatic and spherical aberrations using flint and crown glasses. While the achromatic lens gave a perfectly satisfactory performance inordinary work, something very much better was required for work demandinghighly critical high-power microscopy. Alternative types of glasswith different optical properties were at that time simply not available.
Despite the impossibility of obtaining glasseswith the special properties he needed, Abbe continued his experiments andwas able to test his theory by using specially prepared fluid elementsas lenses. Two such experimental objectives were made at the Zeissworks, and although they proved fully the correctness of Abbe's theorythey were quite unsuitable for commercial production. There the wholematter might have ended had not a Dr Otto Schott learned of Abbe's failureto obtain the glass he needed. Schott was well suited to undertake theexperimental work: he had a good theoretical background and had been trainedm the practical side of glass manufacture in his father's glass factory,which also gave him ready access to the facilities he needed. Ina short while he wrote to Abbe reporting his Success in formulating a newtype of glass with optical properties quite different from those of ordinaryglass. With Abbe's constant encouragement Schott continued his experimentsand produced more new types of glass. Although he was only able tomanufacture small quantities of the new glass there was enough availableto make a prototype lens which demonstrated the possibilities of the newobjectives.
With financial assistance from the Prussiangovernment, and the support of the Zeiss organisation, a glass researchlaboratory-which later expanded into a small glass-making plant-was openedin Jena with Schott in charge. The new Schott glass works and theZeiss organisation worked in close contact, and by the time Abbe had completedhis calculations for the new range of apochromatic objectives adequatequantities of the new glass were available for commercial production tocommence.
Somewhat surprisingly, Abbe's name is perpetuatednot by any of his original contributions, but by a piece of equipment whichhad been in use long before his time-the substage condenser. Abbedevised an efficient condenser with a special mounting, which is stillknown as the Abbe condenser.
Lifelong collaboration with Zeiss
Ernst Abbe, the son of a spinning-mill foremanin the small German town of Eisenach, was born on 23 January 1840. He went to the University of Jena and then to Gottingen, where in 1861he obtained his degree. After posts at Gottingen and Frankfort-am-Mainhe returned in 1863 to a lectureship at Jena. Three years later hewas invited by Carl Zeiss to join the Zeiss organisation as a scientificconsultant, and so began one of the most fruitful of scientific partnerships. Abbe's duties with Zeiss allowed him to retain his university post, andin 1870 he became a professor. Abbe greatly valued his continuedassociation with the university and was always ready to offer promisingyoung scientists posts in the Zeiss organisation, and to encourage Zeissscientists to lecture at the university. In 1875 his close collaborationwith Carl Zeiss was further strengthened when he became a partner in thefirm, and in 1888, on the death of Zeiss, he became its proprietor.
Abbe died on 14 January 1905, a few days beforehis 65th birthday. A most impressive monument to his memory was erectedin Jena and each year is visited by scientists from all over the world.
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