This page contains historical
notes on pioneers of Medical Laboratory Science
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Original paper on Grams's Stain
Original paper on using the Petrie Dish
1861 – 1947
In his long and active life in medical research Sir Almroth Wright made many valuable contributions to the welfare of mankind. Discoverer of a successful method of anti-typhoid inoculation, archpriest of vaccine therapy, tireless researcher into the bacteriology of wound infection: these were but some of the scientific activities of this remarkable man. Among his friends he numbered many of the great figures of his day; famous scientists, politicians, philosopher, lawyers, poets and playwrights, all came to talk and exchange ideas with him.
Though his scientific contributions were of tremendous importance, without his courageous fighting spirit to advance them – often in the face of influential opposition – they would have achieved little. In persuading Lord Kitchener in 1914 to issue an order that no troops were to be posted overseas without anti-typhoid inoculation he probably saved the lives of 125 000 British soldiers, and prevented ten times that many cases of the disease.
Much of the success of Sir Almroth's research was undoubtedly due to his complete mastery of laboratory technique. In those days medical laboratory equipment was in no way comparable with that of modern laboratories but was very modest – usually little more than a bench, microscope, and incubator – few of the essential instruments of modern medical laboratory science having been devised. Wright's requirements were very simple: mainly a supply of glass tubing, plasticine, rubber teats and a Bunsen burner. How this master of technique would view the needs of modern laboratories, with their ever-increasing dependence of highly sophisticated and expensive apparatus, makes interesting speculation.
Technique of the teat and the Capillary
In many of his researches Wright had to develop new techniques – for example in the measurement of phagocytosis, bactericidal power and opsonic index, and in the modification of Widal's test – and he can be said to have introduced micro-methods to bacteriology. In Wright's early days vein puncture was rarely practised to obtain blood specimens, capillary collection being the established method. The many ingenious but simple techniques he evolved were published in book form in 1912 with the title The Technique of the Teat and Capillary Tube. This book became a classic and in 1921, in collaboration with Dr Leonard Colebrook, Wright revised it and a new edition was produced.
It was fortunate that Wright's requirements of equipment were modest. When he was appointed to the post of Pathologist and Bacteriologist to St Mary's Hospital, Paddington, and the Hospital Committee considered they were very fortunate in obtaining the services of what they described as 'so eminent an investigator'. Wright had made it a condition of his acceptance that the equipment of the laboratories should be the best available, but the committee only found it necessary to spend £100 – although later they spent a further £100 on structural alterations to the museum, of which the laboratories formed a part.
While Wright did not invent the rubber teat, he was responsible for its use as an alternative to mouth suction of pipettes. In 1898 he published a paper on the technique of serum diagnosis, and in it he recommended the use of rubber teats for pipette work. Wright suggested two sizes of teat, one with a capacity of 1.5cm³ and a larger one of 2.5cm³. Both were to be made from the best quality soft vulcanised rubber.
Wright's blood capsule
In the years when the Pathological and Bacteriological Laboratory Assistants Association held its diploma examinations, many a candidate was confronted with Wright's blood capsule in the viva room. Wright developed this apparatus for the collection of clotted specimens of capillary blood, and in the first part of this century it enjoyed widespread use.
The capsule could be made in a Bunsen flame, using a short length of 0.5cm diameter glass tube: one end was drawn out into a straight capillary, the other being drawn out into a capillary and bent at an angle of 30 degrees. Both capillary ends were then sealed the centre part of the glass tube forming the capsule, sufficient to contain about 0.4cm³ of blood. For use the capillary ends were cut and the curved capillary applied to the puncture site, when blood could run into the capsule by capillary attraction and siphonage, air escaping by the straight capillary. When collection was complete the capillary ends were sealed with a lighted match if a Bunsen burner was not available.
In these days of disposable apparatus there can be very few who realise the unique virtues of Wright's blood capsule, Sterility was no problem; the straight ended capillary could be used as a pricked; the curved limb was handy to hand on a centrifuge bucked, was a convenient place for the attachment of an identification tag, and allowed the capsule to be stood on the bench. Lastly, the capsule had the property demanded of modern specimen containers – it was disposable.
Wright's powers of invention and manipulative skill extended to all types of capillary, graduated, throttle and automatic pipettes, vaccine bulbs and many other items.
Life and work
Born in Yorkshire in 1861, the son of an Irish clergyman and a Swedish mother, Wright qualified in medicine and surgery at Trinity College Dublin in 1883. In his early years he spent time in Europe working under some of the greatest men of the time, followed by a period in Sydney. Later he returned to England, working in London and Cambridge and it was during this period that he wrote a handbook on the microscope – in one respect unique in that no use was made of mathematical formulae to explain the optical phenomena: Wright relied on diagrams, a practice that did not commend itself to some reviewers of the book.
In 1892 Wright was offered the Chair of Pathology at the Army Medical School, then located at the Royal Victoria Hospital, Netley, where he did most of his work on anti-typhoid inoculation. Despite great efforts, however, he failed to persuade the Army authorities of the need for compulsory inoculation of troops going the Boer War. A scheme of voluntary inoculation was somewhat grudgingly allowed, but only about four per cent of the troops were protected and the result was that the British Forces had nearly 60 000 cases of typhoid, with over 9 000 deaths. After the war an inquiry into the value of anti-typhoid inoculation reported adversely and the Army suspended voluntary inoculation. Wright, like many others, was appalled. He resigned his post at Netley and appealed to the Secretary of State for War for an inquiry. After much committee work it was decided to institute a five-year trial upon troops posted to India. The results fully proved the value of the vaccine and the Army allowed voluntary inoculation to be re-started in 1910.
With the outbreak of the 1914 – 1918 war Wright was determined that the typhoid losses of the Boer War should be avoided. He sought an interview with Lord Kitchener, who had seen during the Boer War the damage that typhoid could do. On Wright's advice Kitchener issued an order that no soldier was to be posted overseas without anti-typhoid inoculation. Later St Mary's Hosp9ital in Paddington turned to vaccine therapy, and the 'Inoculation Department' Wright established there became an international centre. It is something of a paradox that this same department later gave birth to the first of the antibiotics, which, with the 'Sulfa' drugs, largely displaced vaccine therapy.
The Inoculation Department later became the Wright-Fleming Institute with Sir Almroth Wright as its first director. This great man, whose name must rank with those of Paster and Koch, continued to work almost up to the day of his death at the age of 85, in 1947.
NOTE: This historical note has
been abstracted from 'Founders of Medical Laboratory Science' and is provided
for educational interest purposes.
James Lorrain Smith
1862 - 1931
To many older workers in the medical laboratory sciences the name of Professor Lorrain Smith is associated with the once popular Nile blue sulphate method of staining fat in tissue sections. Important though his many investigations on fat and lipoids were, they constituted only one part of his scientific work. Other spheres of medical research in which he made equally important contributions included respiration, body temperature, bacteriology and antiseptics, and he was a widely acknowledged authority on medical education.
Fats and lipoids
In the early 1900s, when Lorrain Smith held the chair of pathology at Manchester, he undertook research into fats and lipoids. This interest he never abandoned, publishing over twenty papers on the subject following his first in 1906. His interest in histochemical methods for the demonstration and differentiation of fat in sections appears to have been stimulated by a chance observation on a section of amyloid liver. This had been stained with gentian violet, and it happened that the whole of the section had not bee completely covered by the cover slip. He noticed that in the uncovered part of the section fat globules had stained with the dye. Subsequent investigation showed this to be due to the action of atmospheric carbon monoxide on the neutral fat, the free fatty acid then combining with the coloured base. He carried out an exhaustive series of trials using very many different dyes. One of the dyes tested, Nile blue sulphate A, appeared to differentiate between neutral fat and fatty acids and from this was evolved his Nile blue sulphate method. Though modifications of this procedure are still employed for certain purposes, subsequent investigation showed that it was impractical to distinguish different kinds of fats by histochemical staining methods.
Working with W Mair, Lorrain Smith also turned
his attention to Wergert's method of staining medullated nerve fibres and
found that by prolonged immersion of the tissue in a strong chrome solution
a similar picture to that obtained by Marchi's process was produced, in
which only degenerated myelin was stained.
Work on respiration
In his early career Lorrain Smith worked in Oxford, where he became associated with John Haldane in a series of blood and respiratory investigations. This association continued in the various posts Lorrain Smith held in the following ten years, first at Cambridge and subsequently at Belfast. One of the most important pieces of work which resulted from the partnership was a paper published in 1900 dealing with the oxygen capacity of blood. In this it was clearly shown that the oxygen capacity of blood was directly proportional to the intensity of its colour. Some years later this finding played a big part in the introduction of Haldane's carboxyhaemoglobin method for the estimation of haemoglobin. This simple visual colorimetric technique remained one of the principle procedures for the clinical estimation of haemoglobin for over four decades. Lorrain Smith also published a new method of preparing culture media and devised two microscope warms stages, one of which was among the first of the electrical types.
Other fields of investigation in which he made important contributions included a series of papers and a report on the Belfast typhoid epidemic of 1898. During the 1914 - 1918 war, along with a small team he investigated the problem of antiseptics for war requirements. The outcome of this work was the appearances of the preparation known as 'Eusol', once very widely used. During the same period he also took part in an investigation of the pathology of trench frost bite, which resulted in a number of practical preventive measures.
Formation of PBLAA
Of the many eminent pathologists who gave their support and help in the formation of the Pathological & Bacteriological Laboratory Assistants Association (PBLAA), the predecessor of the Institute of Medical Laboratory Sciences, none played a more significant role than Professor Lorrain Smith. Albert Norman, founder of the PBLAA, was then Lorrain Smith's personal laboratory assistant and he enjoyed the full support and encouragement of his chief in the project. Professor Lorrain Smith undertook to bring the scheme to the official notice of the Pathological Society of Great Britain and Ireland, itself only five years old. To have any chance of success it was essential that the new association should have the blessing and support of this influential body.
The aims and objectives of the Association
were at first widely misunderstood, and the very idea received with considerable
suspicion. By no means all pathologists welcomed its formation.
At the Liverpool meeting of the Pathological Society in 1912 sympathy for
the project was recorded, however, and willingness to assist in its objectives
expressed. the new organisation needed all the support and help it
could get; the initial membership was a mere 24, which by the end of the
first year had risen to only 84. During the difficult years afters
its formation the PBLAA enjoyed the support of Professor Lorrain Smith
and the Pathological Society. In 1913 he became its first President,
an office which he held until 1915 when he was followed by the distinguished
figure of Sir G Sims Woodhead.
Founder of the Pathological Society
James Lorrain Smith was born on 21 August 1862 in the small Dumfriesshire village of Half-Morton, where his father was the Free Church minister. He graduated at Edinburgh in 1889 and , shortly after qualification, went to Oxford to work under Sir John Burdon-Senderson, followed by a spell at Cambridge in Charles Ray laboratory. He also spent periods of study in the laboratories of Christian Bohr in Copenhagen and Friedrich Von Recklinghausen in Strasbourg.
In 1894 Lorrain Smith was appointed lecturer in pathology in Belfast and seven years later, when a chair was created, became professor. His next move was to Manchester as professor of pathology, and during this time he formed one of the small groups of pathologists which was responsible for the foundation of the Pathological Society. In 1912 he returned to Edinburgh as professor of pathology.
Though his scientific work never ceased, during
the later years of his career much of this time was devoted to academic
and public work. Early in 1931 he suffered a sudden severe illness
from which he never fully recovered, and he died on 18 April.
Robert Koch is rightly regarded as the founder of clinical bacteriology. Not only is he justly famed for his discovery of the causative organisms of tuberculosis and cholera, but also for his work on anthrax. The techniques he introduced, made possible the advances which have resulted in modern bacteriology. His work covered a wide field, much of it devoted to establishing the role of bacteria in disease. He also first described the phenomenon of phagocytosis, but failed to grasp its true significance and four years later the great Elie Metchnikoff published his famous Theory of Phagocytosis.
Casimir Davaine, a leading parasitologist of the time, had already identified the causative organism of anthrax, but his work left problems of epidemiology unsolved. Koch's contribution was in demonstrating spore formation in this organism, and its part in the life cycle.
At the University of Breslau Koch, then an unknown provincial doctor, demonstrated his successful experiments on anthrax to Professor Ferdinand Cohn. He was able to show that the short rod-like organism, recovered from the blood of an infected animal and grown in culture, produced a long chain of bacilli in each of which a spore appeared. This was the first complete life cycle of a bacterium to be described.
In the 1880s tuberculosis was the great 'killer' of civilised communities. It is recorded that something like fifteen per cent of deaths were due to this one disease. Some fifteen years previously, a French army doctor, Jean Antoine Villemin, in a brilliant piece of experimental work, had proved the disease to be infectious due to a specific, but unknown, agent. Koch, in his research into tuberculosis, succeeded in discovering the causative organism - the tubercle bacillus, which was at first known as 'the bacillus of Koch'.
None of the existing methods of staining was satisfactory for demonstrating is bacillus but after much trial and error Koch evolved a successful procedure. His method was subsequently much modified by others - notably Ehrlich, Ziehl, and Neelsen - and is now known as Zichl-Neelsen's method.
Koch was also successful in growing the tubercle bacillus on artificial culture medium: for this he devised a special medium using blood solidified by heat at 65° C. By what later became regarded as a classical masterpiece of research, Koch conclusively proved that the bacillus he had isolated was the cause of the disease. A preliminary report of his findings appeared in the Berliner klinische Wochenschrift in 1882, and at about the same time he presented a paper on his findings at a meeting of the Physiological Society of Berlin. Two years later a complete statement of all his work on tuberculosis was published in Mittheilungen aus dem kaiserlichen Gesundheitsamte.
Later in life Koch was to return to the study of tuberculosis and search for a protective or curative substance - a project that lead him into grave error. World medical opinion was very interested when, at the Berlin International Medical Congress in 1890, Koch announced that he had found a cure for tuberculosis: tuberculin. As a valuable diagnostic aid there was little doubt about the value of tuberculin, but almost from the start grave doubts were expressed about its therapeutic value - doubts that were fed by the scanty evidence Koch produced in support of his claims.
The subsequent failure of tuberculin as the great specific remedy for tuberculosis was a humiliating experience and brought sorrow to Koch. It caused immense damage to his reputation and remains a blot on a wonderful record. There has been much speculation why Koch, an experienced and careful worker, should have made public his claims before he had completed adequate investigations. It must be remembered, however, that the time was one of considerable international rivalry in bacteriological research; it is more than possible that official pressure was put on him to produce something outstanding at the Berlin Congress .
In 1883 a severe outbreak of cholera occurred in Egypt, with deaths mounting at the rate of 5000 a week. The Egyptians appealed to the two leading schools of bacteriology for help, those of Louis Pasteur in Paris and Robert Koch in Berlin. Both the French and the German government sent teams. Pasteur, through illness, was unable to lead the French team but sent one of his most able assistants, the twenty-seven year old Louis Thuillier, who was to die of the disease in Alexandria.
The German team led by Koch set up their laboratories in the Greek Hospital of Alexandria. Intensive work resulted in the finding of a strange comma-shaped micro-organism in the intestines and excreta of people suffering from the disease. Koch was reasonably confident that he had isolated the causative organism, but by now the Egyptian epidemic was ending so he moved to Calcutta where the disease was endemic. Here he quickly proved the correctness of his findings, and in 1884 was able to announce to the world that the causative organism was the Vibrio cholerae.
Contribution to technique
There is no doubt that Koch, by his technique for obtaining pure cultures, made the greatest single contribution to the development of bacteriology. He realised the limitations of fluid media and, while solid media in the forms of the cut surfaces of boiled potatoes, and coagulated blood, were being used, Koch's search was for a solidifying agent which could be added to the existing fluid media. First he turned to the obvious agent, gelatin, but quickly recognised its disadvantages. Agar was the answer, its use being suggested by old student, Walther Hesse.
Credit for the modern technique of making and staining smears on slides appears to be due to Koch, though he seems to have preferred fixation by fluid fixatives rather than flaming. He was also largely responsible for the introduction into bacteriology of the Abbe condenser and Stephenson's highpower oil immersion lens.
Koch investigated the efficiency of disinfectants and sterilising processes; Koch steam steriliser was at one time an essential part of the equipment every bacteriological laboratory. He established certain criteria for the identification of a disease's causative organism, and these became known as Koch's postulates'.
Koch was born at Klausthal in Hanover on 11 December 1843, his father being a mining engineer of some standing. He studied medicine at the University of Gottingen, obtaining his doctor's degree in 1866 and his first post appears to have been that of assistant in the General Hospital, Hamburg. Like many of his contemporaries he enlisted as a surgeon in the German army in the Franco-Prussian War of 1870.
After the war Koch obtained the post of Kreis-physicus, or district physician, in the town of Wollstein in East Germany. Despite the isolation his position brought from research centres, Koch pursued his study of anthrax. At his own expense he fitted out a small laboratory, equipped with such essential items as a good microscope, microtome, and incubator. The delightful story related of how his wife, Emly, bought the microscope with money painfully saved from her house-keeping allowance, carefully concealing the money in an old beer mug. While this story may be true, the impression of poverty it suggests is not. Koch was fortunate in escaping the frugal life which was the fate of so many of his colleagues, at least in their early days.
It was at Wollstein that Koch carried out his
work on anthrax, the fame of which brought him an appointment at the Reichsgesundheitamt
in Berlin, there he developed new methods of bacterial culture and made
the discovery of the tubercle bacillus in 1882. The following year
saw him in Egypt and India, head of the German Cholera Commission which
resulted in the discovery of the cholera vibrio. He returned to Germany
to be feted as a national hero, awarded 100 000 marks by the German government.
and honoured by Kaiser Wilhelm 1.
In 1885 Koch became professor of hygiene and bacteriology in the University of Berlin. Here he organised the first courses in practical bacteriology and his assistants and students included many who, like Gafflky, Loeffler, Von Behring, Pfeiffer, Ehrlich, Gartner, and Wassermann, were later to become household words in the bacteriological field. Six years later Koch became director of the newly built Institut fur Infectionskrankheiten (Institute of Infectious Diseases) in Berlin. He was then the principal medical adviser to he German Colonial Office and spent a good deal of time in the tropics.
He appears to have developed a liking for travel and in 1904, at the age of 61, resigned his high office in Berlin and devoted himself to the study of tropical disease. While he worked mainly in Tanganyika on sleeping sickness, he also investigated rinderpest of cattle in British South Africa, plague in India, and malaria in Java, Sumatra, and Malaya. In 1905 Koch was awarded a Nobel Prize.
Despite the many honours and the fame which rewarded his work, Koch's latter years were saddened by the failure of tuberculin and by the domestic upheavals of his private life. He died of cardiac failure in 1910 and his ashes were deposited in a special room at the institute he founded in Berlin, together with the many medals and orders he had received.
NOTE: This historical note has
been abstracted from 'Founders of Medical Laboratory Science" and is provided
for educational interest purposes.
1876 - 1928
1876 - 1928
This brilliant Japanese scientist, whose work at the Rockefeller Institute had earned him an international reputation, was the victim of one of the most poignant tragedies of scientific research. Noguchi's work in South America had led him to believe that a leptospiral infection was the elusive causative agent of yellow fever. There was a great deal of support for the case he presented, until other investigations of the disease in West Africa failed to confirm his findings and it was announced that a filterable virus was the cause of the fever. Noguchi went to Africa hoping to verify his results. He was unsuccessful and, during his last investigation suffered the bitter experience of discovering that the theory on which all his work on yellow fever had been founded was fundamentally wrong. He met his death from the disease he had hoped to conquer; a sad and depressed man, he had confided to a friend 'It is the sunset of Noguchi'.
Cultivation of treponema pallidum
Much of Noguchi's early work had been concerned with Treponema pallidum and he demonstrated the presence of this organism in the brain tissue of 'paralytics', thus supplying evidence for the cause of the condition. In 1911 he was the first to obtain pure culture medium consisting of a semi-solid mixture of agar and hydrocele fluid, to which a small portion of rabbit kidney tissue was added and the surface of the culture then covered by a layer of paraffin oil. Also he prepared an extract from cultures of Treponema pallidum which he called 'luetin'. This could be used in a diagnostic procedure for suspected syphilis, an intradermal injection of 'luetin' into a patient producing a local reaction. This test, however, was never widely used since more reliable and convenient methods were available. Noguchi also produced a variation of the Wasserman reaction which, at one time, found considerable favour. In his rather radical modification of the original technique he used an anti-human haemolytic serum and a specially prepared antigen.
Noguchi had widespread interests in medical research and he investigated many problems including trachoma, anterior poliomyelitis, rabies, hog-cholera, the virus of herpes, and Rocky Mountain fever. Prior to his research on Treponema pallidum he had spent a considerable time studying snake venoms and, with Simon Flexner, had published an exhaustive work on the relationship of these to haemolysis, bacteriolysis, and toxicity. He was also the author of an important monograph on snake venom, published by the Carnegie Institute.
Work on yellow fever
Noguchi was not the first to search for, and claim success in finding, the infective agent (carried by the mosquito) which transmitted yellow fever.
In 1881 Carlos Finlay of Havana began his long battle to incriminate the mosquito, and the subsequent investigations of Walter Reed and his team during the American-Spanish war some twenty years later fully confirmed his theory. A number of researchers claimed to have found the cause of the fever including Sternberg in 1888 with his Bacillus X, and the Italian bacteriologist Sanarelli with his Bacillus icteroides, but the search continued until Adrian Stokes finally found the causative agent in 1927.
Noguchi had entered the field in 1918 when he published the results of his work in South America. He had inoculated guinea pigs with blood from cases of what was considered to be yellow fever, and in these animals he produced a condition similar to that disease and demonstrated a leptospira infection. He named the organism he isolated Leptospira icteroides, and the results of a comprehensive series of serological and animal test appeared to fully support his claim. There is evidence to suggest that the initial cases supplied to Noguchi included patients with Weil's disease, and this may well have been the factor which led him onto a false trail of work lasting ten years.
In 1927 members of the Rockefeller West African Yellow Fever Commission announced that the long-sought aetiological factor was a filterable virus. Although in a poor state of health - he had suffered from diabetes for sometime - Noguchi left for Accra to investigate the rival claim Sadly for Nuguchi his research in the laboratories of the Ridge Hospital failed to produce evidence the Leptospira icteroides was the causative agent and he had to accept that either the West African and South American yellow fevers were different diseases, or that there was a basic error in his work.
Pupil of Kitasato
Hideyo Noguchi was born on 24 November 1876 at Inawashiro Yama, Fukushima, Japan where there is now a memorial house in his name. He graduated in medicine at Tokyo University in 1897, and for two years was assistant to Shibasaburo Kitasato at the Tokyo Institute for Infectious Disease. In 1901 he went to America, working first as an assistant with Simon Flexner at the University of Pennsylvania and later at the Carnegie Institute in Washington. After spending a year at the State Serum Institute in Copenhagen he returned to a post at the Rockefeller Institute, with which he was associated for the rest of his life.
Noguchi died of yellow fever at the Ridge Hospital, Accra, on 21 May 1928, one of the many investigators to lose their lives in the conquest of this disease. His remains are buried in Woodrome cemetery, New York. Noguchi's memory is perpetuated in Accra by a bronze bust in a commemorative garden situated some fifty yards form his old laboratory, and his homeland, Japan, honoured his memory in 1949 by issuing a postage stamp bearing his portrait.
NOTE: This historical note has
been abstracted from 'Founders of Medical Laboratory Science' and is provided
for educational interest purposes.
1801 - 1858
1801 - 1858
Johannes Müller was one of the great natural scientists of the first half of the 19th century. He was an eminent contributor to a wide range of scientific, subjects including biology, human anatomy, physiology, embryology, histology, and pathology. In keeping with the contemporary practice of European universities his last appointment - as professor at Berlin, Germany's leading university - covered the three disciplines of anatomy, physiology and pathology. This was the last occasion when the three subjects were combined under one professor at that university.
Müller's work had a tremendous influence on German medicine. He was responsible for a completely new approach to scientific investigation. Before his influence brought about a more critical and logical approach, German scientific thought tended a surround itself with a certain air of mysticism. Despite many teaching commitments and administrative duties, Müller energetically maintained his original work and, while his professional life was a comparatively show one (stretching over little more than a quarter of a century) his tireless enthusiasm resulted in the publication of over 200 scientific papers and several test books. His Handbuch der Physiologie des Menschen, published in the 1830s, became a standard text and he also founded a journal, Müller's archiv.
Work in histology and pathology
Unlike the botanist, the early workers in the fields of histology and pathology somewhat surprisingly made negligible use of the microscope; in fact, many of them appear to have distrusted observations made with it. In this Müller was a major and influential exception.
Although the microscope was still at a relatively elementary stage of optical development, achromatic lenses began to appear in the 1830s and many of the instruments then available were capable of considerably extending the former simple microscopic inspection of tissues. Müller's use of the microscope was ably supported by a contemporary, Johann Evangelista Purkinje, Professor of Physiology and Pathology at the University of Breslau and later at Prague; together the two men were largely responsible for the groundwork of modern histology.
Müller gave a complete description of
the glandular and cartilaginous tissues, and grouped various cells under
the general designation of 'connective tissues'. In 1838 he published
a major work on tumours, and made extensive use of the microscope in his
research for the book.
Müller is often credited with the introduction of potassium dichromate as a histology fixing and hardening reagent, and the potassium dichromate and sodium sulphate mixture used later as the basis for such valuable fixatives as Zenker's and Helly's fluids still bears his name. Others, however - in particular Purkinje - are known to have been using potassium dichromate solutions at about the same time. Improvements in the microscope, and its increased use in medical research, created a need for improved methods of specimen preparation and by the 1830s reagents such as potassium dichromate, glacial acetic acid, osmium tetroxide, and Canada balsam were being used by many medical microscopists.
Müller did not limit his studies to the fields of histology and pathology. His name as an embryologist is perpetuated by the duct he discovered in the early fetus, while in physiology his studies included an investigation of specific nerve properties and the discovery of the lymph hearts of the frog. He also carried out experiments on voice production and on blood coagulation, and undertook an explanation of the function of the bristle cells in the internal ear. His many pupils included such well known figures as Theodor Schwann, originator of the cell theory; Jakob Henle, one of the greatest of early histologists; Robert Hermann von Helmholtz, inventor of the ophthalmoscope; and the great Rudolf Virchow, a pioneer in the study of cellular pathology.
Müller lacked the educational advantages of a wealthy family background. His father was a shoemaker who ran a small business in the city of Coblenz on the Rhine, which was then under French control. Müller was born there in 1801 and was educated locally. He proved to be an exceptional scholar and his father was persuaded to allow him to go to university rather than take up the family trade. At the age of 18 Müller left home to enter the University of Bonn. At first he appears to have studied theology, but soon changed to medicine.
As a student Müller fully justified that faith of his first teachers; his work was recognised as outstanding and an essay entitled Respiration of the Foetus gained him a prize. After three years at Bonn he graduated and was awarded a scholarship which enabled him to undertake a year's further study of anatomy and physiology in Berlin. In 1824 he returned to Bonn to the post of privat-docent and, two years later, was made professor. In the 1830s he was appointed to the professorship at Berlin, where he also served as director of the Anatomical School of Medicine.
Müller was never a particularly happy
man; temperamentally he was moody and withdrawn, and the later years of
his life were marked by unhappy events. The revolution of 1848 had
a profound effect on him; by nature he had little interest in politics,
but the situation forced him to take sides, and for a time his scientific
work suffered. Finally a personal tragedy occurred.
During a trip to Norway to collect ocean fauna
the ship in which he was travelling was wrecked. Müller narrowly
escaped but many of his fellow passengers were drowned, including a young
assistant who was travelling with him. Müller appears to have
held himself responsible for the young man's death and he never completely
recovered from the tragic event. He died in 1858 at the comparatively
early age of 57.
NOTE: This historical note has
been abstracted from 'Founders of Medical Laboratory Science' and is provided
for educational interest purposes.
1851 - 1902
In spring 1900 a virulent yellow fever epidemic was raging in Havana, which was at the time filled with American soldiers engaged in the Spanish-American War. The American forces had already been fighting in Cuba for two years and had suffered heavy losses from typhoid and other diseases. However, when the yellow fever epidemic hit them the casualty list became little short of disastrous. Although the Americans had their share of battle casualties these were of little significance compared with the thousands of deaths from yellow fever. In an effort to avoid what could easily have become a catastrophe the US authorities took every possible hygiene measure, but without any appreciable success.
The US yellow fever commission
While the terrible death toll continued the American authorities appointed a Yellow Fever Commission with instructions to find the cause of the disease and the means of preventing infection. This was certainly a weighty responsibility for the Commission and, what was more to the point, quick results were wanted as scores of young American soldiers were dying every day. The Commission's director was Walter Reed, then a 49 year old major in the US Army Medical Corps, who had already worked in Cuba on typhoid.
Although he had spent all his professional life as an army doctor and much of his time serving in remote military posts in the United States, Reed had also had periods of further study at Johns Hopkins University, where he came under the influence of the great American bacteriologist Professor Welch. He had also held important posts on the staff of the medical division of the Military Academy in Washington.
Reed was assisted by three other members of
the Commission; James Carroll, and English-born bacteriologist; Jesse Lazear,
an entomologist trained in Europe; and a Cuban pathologist name Aristide
Agramonte. Carroll had worked previously with Reed as an assistant
US Army surgeon when, in 1897, they had both investigated Sanarelli's claim
to have found the causative organism of yellow fever, which he called Bacillus
icteroides. Reed and Carroll had proved the claim to be false, identifying
the organism as the hog cholera bacillus. Both Lazear and Agramonte
were also members of the US Medical Corps. Agramonte had already
survived an attack of yellow fever an done of the few things known about
the disease was that recovery from the fever conferred immunity to further
Initially the Commission's approach to the problem was a strictly orthodox bacteriological one - a search for the causative organism. There were many yellow fever patients in the Las Animas Hospital where they were working, and the Commission instigated a complete and painstaking bacteriological examination. When victims of the disease died, as all too often they did, an autopsy was carried out with meticulous care. However, after weeks of intensive work the search for a causative organism was completely unrewarding and the Commission found itself back where it had started, with no tangible achievements whatsoever.
A curious observation
It then came to Reed's knowledge that a convict who had been under strict security for five weeks had contracted the disease, his only human contacts being eight other prisoners and the guards, all of whom had remained healthy.
Reed also notice that nurses who were non-immune rarely contracted the disease, yet they were in daily contact with yellow fever patients. Surely, if the disease was caused by conventional microbe these people would be at great risk and many should have fallen victims. Some other factor must therefore be carrying the infective agent - possibly an infected insect.
All this caused Reed to remember the work of the Cuban, Carlos Finlay, who had spent nearly twenty years expounding his theory that certain type of mosquito was responsible for transmitting in the infection. Major Reed with members of his commission, paid a visit to the old Cuban doctor who was then 77 years of age. Finlay received his important guests with the greatest courtesy, taking pains to explain in detail his theory of the role of the mosquito in the transmission of yellow fever, but freely admitting his lack of experimental evidence. Reed and the members of the Commission, however, were deeply impressed; this possibility must be thoroughly investigated and the Commission had all the resources which Finlay had lacked to carry out the necessary experiments. They gladly accepted Finlay's offer of some mosquito eggs of the species which he considered responsible for transmission of the disease, and from these they were able to breed stock of mosquitos for their investigations.
The story of the Commission's work in proving the truth of Finlay's theory has become part of the history of tropical medicine. The construction of a mosquito-proof station called Camp Lazear was a tribute to their colleague, who had by this time fallen victim to the disease.
Heroic volunteers, led by Dr Carroll, allowed themselves to be bitten by infected mosquitos - and equally heroic volunteers proved that bedding and clothing, grossly contaminated with excreta by victims of the disease were non-infective. Walther Reed, although a kindly and gentle man, did not hesitate to use human volunteers in his work on yellow fever, some of whom paid for their courage with their lives. The employment of human volunteers was justified on the grounds both of urgency and the unavailability of suitable experimental animals. In fact it was not until 1928 that Adrian Stokes reported the successful experimental infection of the rhesus monkey, and two years later the Max Theiler discovered that white mice were susceptible to intracerebral inoculation of the virus.
As a result of its intensive study the Commission was able to announce at the Hygiene Congress in Indianapolis that the mosquito Aedes aegypti was the intermediate host of the infective agent responsible for yellow fever. though this work formed only the starting point for complete control of the disease, and left many important questions still to be answered, its effects were immediate. Within a year preventive measures instituted by William Crawford Gorgas had freed Havana from the disease, and subsequently made possible the construction of the Panama Canal.
Soldier and scientist
Walter Reed was born on 13 September 1851 at Harrisonburg, Virginia, the youngest child of a family of five whose father was a Methodist minister. Reed studied medicine at the University of Virginia, obtaining his MD at the age of 18 - the youngest medical graduate then known. He later moved to Bellevue Medical College in New York, where he obtained a second degree in medicine. In 1874 he decided to become an army surgeon and obtained the post of assistant surgeon in the US Medical Corps with the rank of first lieutenant. After some six years of service in various US Army posts he was promoted to captain. This promotion brought him to Baltimore and gave him the opportunity to undertake further study at Johns Hopkins University.
In 1893 Reed was appointed curator of the Army
Medical Museum and Professor of Bacteriology and Clinical Microscopy at
the Military Academy in Washington. With the outbreak of the Spanish-American
War in 1898 he applied for active service and was posted for duty in Cuba,
first to study typhoid and subsequently as Director of Yellow Fever Commission.
His death, on 2 November 1902 at the early age of 51, prevented him seeing
the full success of his work and enjoying the honours he so justly deserved.
He was buried in the National Cemetery at Arlington.
|Friedrich von Recklinghausen
1833 - 1910
The last quarter of the 19th century was a
period particularly rich in medical scientists. While many of these
became famous through their discoveries in the new and rapidly developing
science of medical bacteriology, the older discipline of pathological anatomy,
particularly in the university centres of Germany, also produced its share
of famous figures. Friedrich von Recklinghausen was among the most
brilliant of these, his career culminating in the post of Professor of
Pathology at Strasbourg University. His reputation as a teacher attracted
students not only from all over Germany but from other parts of the world
also. Von Recklinghausen was no narrow specialist; he worked in many
fields of pathology and his name is perpetuated in the condition known
as von Recklinghausen's disease, or neurofibromatosis.
Von Recklinghausen's disease
In 1882 von Recklinghausen published his description of neurofibromatosis in a paper which became a classic. Robert William Smith was the first person to describe the condition accurately in the medical literature some thirty years previously, but von Recklinghausen's paper added considerable new knowledge of the condition and its pathology, and subsequently the disease was named after him.
Von Recklinghausen also developed a deep interest in, and was an authority on, the pathology of disease of the bones. In 1892 he published a paper describing generalised osteitis fibrosa, a disease which some decades later was found to be caused by a parathyroid tumour. At one time this disease was described as von Recklinghausen's disease of the bone but the association of his name with two totally different diseases caused confusion, and its use in describing the second condition was discontinued.
Apart from his many contributions to the pathology
of bone disease von Recklinghausen made extensive studies of many other
subjects, including embolism, infarction, and thrombosis. He was
also the first to describe fatty and hyaline degeneration of muscle and
in a paper published in 1889 he introduced the word haemochromatosis.
Silver impregnation of tissues
Von Recklinghausen was not only a great experimental pathologist and authority on pathological anatomy: he was also a gifted technologist who introduces several technical procedures and instruments. He was one of the first to use a metallic impregnation method in the preparation of tissue for microscopy. As early as 1862 he described the use of a weak solution of silver impregnation method for the staining of nervous tissue. During his investigation of inflammation of the cornea and of the motility of cell he invented a form of moist chamber.
Von Recklinghausen was also interested in the development of blood cells, and extensively studied the morphology and staining reactions of leucocytes. He was one of several investigators who drew attention to the amoeboid movement of leucocytes in the bloodstream, and in 1885 published a paper which extended and supported Huber's work on the relationship of chloroma and leukaemia.
A great teacher
Friedrich Daniel von Recklinghausen was born in 1883, in the town of Gutersloh in the industrial province of Westphalia. He went to Berlin for his medical education and obtained his degree in 1855. In Berlin he was impressed by the world famous Rudolf Virchow and, after graduation, spent six years as his assistant at the Pathological Institute of the Charite Hospital in Berlin, of which Virchow was the director.
In 1865 von Recklinghausen was appointed Professor
of Pathological Anatomy at the University of Konigsberg and a year later
moved to a similar position at Wurzburg. When the new University
of Strasbourg was founded in 1872 he made his last move, to take up the
appointment of Professor of Pathology there, a post he was to retain until
he retired some thirty years later. During his time in Strasbourg
his work and reputation as a teacher made it one of the greatest centres
of pathology in Europe. Von Recklinghausen is said to have been a
man of the highest personal character and a kindly soul, loved by all with
whom he came in contact. After his retirement in 1906 he continued
to live in Strasbourg, the city he had come to know so well and love, until
his death in 1910 at the age of 77.
NOTE: The historical notes above
have been abstracted out of 'Founders of Medical Laboratory Science' and
are provided for educational interest purposes.
Presented here is the first report of the bacteriological staining method most widely used today. As first devised by Gram, the method was useful in staining bacteria in tissue sections. In his time this was an important discovery, because studies of the pathogenesis of different species of bacteria was just in its infancy. The first of Koch’s postulates was that the suspected causal organism should always be found in association with the disease. However, this presupposed a method for staining the minute bacteria in lesions so that they should be adequately visualised. Because of the fact that many bacteria exhibit the peculiar staining reaction, which Gram describes here, it was possible to detect them much easier with his method.
For many years the main use of the Gram stain has been to differentiate species of bacteria. In the present paper, Gram describes several organisms that were not stained by his technique. We would call these Gram-negative, and the number of Gram-negative bacteria is probably larger than the number of Gram-positive bacteria. The Gram stain is one of the first procedures learned by beginning bacteriology students and is one of the first procedures carried out in any laboratory where bacteria are being identified. Its importance to bacterial taxonomy is therefore obvious.
The mechanism of the Gram stain is still a partial mystery. As Gram himself noted, the iodine-potassium iodide solution is essential in the reaction. We know that this solution must follow, and not precede, the gentian violet. We know that the iodine and the gentian violet forma a complex inside the cell (Gram also noted this complex formation) which is insoluble in water but is soluble in alcohol. Apparently Gram-positive bacteria are those which are able in some way to keep the alcohol from reaching this insoluble complex. We know that the Gram stain is not an all or nothing phenomenon, but that quantitative variations in Gram-positively exist between different species, and within the same species during different parts of the growth cycle or under different environmental conditions. We know that only intact cells are Gram-positive, so that cells which are even gently broken become Gram-negative. We know that bacterial protoplasts, devoid of cell wall, are still Gram-positive, indicating that it is probably the semipermeable membrane, which is somehow involved in the reaction. Finally, we know that Gram-positively is restricted almost exclusively to the bacteria, with only a few other groups, such as the yeasts, exhibiting this reaction. We can truly say that the implications of Gram’s discovery have been widespread.
Comment by Professor Thomas D. Brock, University of Wisconsin, 1961. Extracted from Milestones in Microbiology – printed by the American Society for Microbiology 1961, 1975.
Grams original article translated by Professor
Gram,C.1884 Ueber die isolirte Färbung der Schizomycetin in Schnitt-und Trockenpräparaten. Fortschritte der Medicin, Vol. 2, pages 185-189
The differential staining method of Koch and Ehrlich for tubercle bacilli gives very excellent results either with or without counter-staining, since the bacilli stand out very clearly due to the contrast effect.
It would be very desirable if a similar method for the differential staining of other Schizomycetes were available which could be used routinely by the microscopist. My studies as associate of Herr Dr Friedländer in the morgue of the city hospital in Berlin have attempted to demonstrate cocci in tissue sections of lungs of those who have died of pneumonia as well as in experimental animals. As Friedländer has already mentioned briefly in his paper on the micrococci of pneumonia, I have discovered by experimentation a procedure for the differential staining of pneumococci. In my procedure the nucleus and other tissue elements remain unstained, while the cocci are strongly stained. This makes them much easier to locate than previously, since in ordinary preparations from pneumonia patients, where such a large amount of exudate occurs, they are impossible to see.
Further studies on the usefulness of this method for other Schizomycetes has gradually shown that this method is an almost general method for all tissue sections and dried preparations. For staining the ordinary anilinegentain violet solution of Ehrlich is used. The appropriate sections must be carried up to absolute alcohol and taken from this directly into the dye solution. They should remain in the dye for 1 – 3 minutes (except tubercle bacilli, which should remain as usual 12 – 24 hours). Then they are placed in a solution of iodine-potassium iodide in water (iodine 1.0 part, potassium iodide 2.0 parts, water 300.0 parts) with or without a light rinse with alcohol and allowed to remain there for 1 – 3 minutes. During this time, a precipitate forms, and the previously dark blue-violet sections now become dark purple – red. They are then placed in absolute alcohol until they are completely decolourised. It is well to change the alcohol once or twice during this step. Then the sections are cleared as usual in clove oil, in which the rest of the dye is dissolved. The nucleus and fundamental tissue is stained only a light yellow (from the iodine) while the Schizomycetes, if any are present in the preparation, are an intense blue (often almost black). The intensity of the staining has not been equalled by any of the current staining methods. This presents another great advantage of out method. It is possible after the decolourisation in alcohol to place the sections for a moment in a weak solution of bismarck brown or vesuvine, and then dehydrate again with alcohol, in order to achieve a counterstain. The nucleus will appear brown, while the Schizomycetes will remain blue.
In this way it is possible to prepare doubly stained preparations that are just as excellent as those of the tubercle bacillus made after the method of Koch and Ehrlich. Permanent preparations in Canada balsam-xylene or gelatine-glycerol remain unchanged after four months. This method is quick and easy. The whole procedure takes only a quarter-hour, and the preparations can remain many days in clove oil without the Schizomycetes cell becoming decolourised.
It is also useful for dried preparations. It is performed exactly as for tissue sections, except that one treats the cover slip just like a section.
I have tried many times different aniline dye (rubine-aniline, fuchsinaniline and simple gentian violet solution), but without success. In addition, tincture of iodine or potassium iodide solution, as opposed to iodine potassium iodide solution, are also ineffective, since the Schizomycetes then are also decolourised. When the sections are treated with water or dilute alcohol, the results are variable.
I) After iodine treatment, the following forms of Schizomycetes are not decolourised by alcohol.
a) The coccus of bronchial pneumonia (19 cases)
b) Pyogenic Schizomycetes ( 9 cases)
c) Cocci of a liver abscess (1 case)
d) Cocci and small bacilli in circumscriptive infiltration of the lungs (1 case)
e) Cocci of osteomyelitis (2 cases)
f) Cocci of suppurative arthritis following scarlet fever (1 case)
g) Cocci of suppurative nephritis following cystitis (3 cases)
h) Cocci of multiple brain abscesses following empyema (2 cases)
i) Cocci of erysipelas (1 case)
j) Tubercle bacilli (5 cases)
k) Anthrax bacilli (3 cases) (in mice)
l) Putrefactive Schizomycetes (bacilli and cocci)
II) The following forms of Schizomycetes are
decolourised in alcohol after iodine treatment.
a) 1 case of bronchial pneumonia with cocci that formed capsules
b) 1 case of bronchial pneumonia with cocci that did not form capsules.
c) Typhoid bacilli (5 cases) are decolourised either with or without iodine treatment very
easily by alcohol. I have attempted to leave the sections in the dye for as long as 24 hours
but without any better results.
I would like to make one closing remark. The behaviour of the cell nucleus and the Schizomycetes to aniline dyes in other methods is almost identical, where as with the present staining method a very distinct difference is visible.
Studies on Schizomycetes have been significantly improved by the use of this method. It is because of this that I publish my results, although I am well aware that they are brief and with many gaps. It is to be hoped that this method will also be useful in the hands of other workers.
Editor’s note I would like to testify that
I have found the gram method to be one of the best and for many cases the
best method which I have ever used for staining Schizomycetes.
A minor modification of plating technique of Koch
1887 * R. J. Petri
Petri, R. J. 1887. Eine kleine Modification des Koch’schen Plattenverfahrens. Centralblatt für Bacteriologie und Parasitenkunde, Vol 1, pages 279 – 280 .
In order to perform the gelatin plate technique of Koch, it is necessary to have a special horizontal pouring apparatus. The poured plates are then placed over one another in layers on small glass shelves in a large bell jar. In many cases it would be desirable to carry out the procedure with less equipment, especially without the pouring apparatus. Since the first of the year I have been using flat double dishes of 10 – 11 cm in diameter and 1 – 1.5 cm high. The upper dish serves as a lid as usual and has a somewhat larger diameter. These dishes are sterilised by dry heat as usual and after cooling the nutrient gelatin containing the inoculum is poured in. The upper lid is lifted only slightly and used as a shield while the tube containing the gelatin, its edge previously flamed and cooled in the usual manner, is emptied into the bottom of the dish. Under these conditions contamination from airborne germs rarely occurs. The poured layer of gelatin soon hardens into a layer several millimetres thick, which can be kept and observed for a long time because of the protecting upper lid. In studies of soil samples, and , earth and similar substances, it is advantageous to place the material in the dish and then pour the liquid gelatin over it. The material is well mixed with the gelatin by rotating the dish with short, intermittent movements. With the dimension give, every spot on the gelatin surface is accessible with the low power microscope. Only when high power lenses are used is the area at the edge of the dish no longer accessible. The gelatin dries in these dishes quite slowly. They can be kept moist longer in 5 – 6 dishes are placed on top of one another on a disc of moist filter paper in a flat dish over which a bell jar is inverted. These dishes can be especially recommended for agar-agar plates, since agar-agar sticks poorly to simple glass plates unless special means are used. In addition, it is quite simple to count the colonies that have grown on the plates. The upper lid is replaced by a glass plate that has etched on it squares of known area. The colonies are then counted against a black background using a magnifier. The total area of the plate can be calculated from the diameter.
We have here the first description of the Petri dish, a simple yet effective device for culturing microorganisms on solid media. The original idea of Petri has not been improved upon to this day, and in bacteriology laboratories all over the world dishes are used of almost the identical features as those first described by Petri. The Petri dish is such a simple idea that if Petri had not thought of it, someone else probably would have conceived of it later. But because of its great usefulness and universality, its first description warrants inclusion in the present collection.
We also have an interesting sidelight in this
article on the procedures used for viable counting. The methods did
not differ much from those we use today, except that in the time of Koch
and Petri, many more viable cells were put in a plate, resulting in a larger
number of colonies and overcrowding. Because of statistical considerations,
we de not use such large numbers of cells, but dilute our samples until
there are between 30 and 300 viable colonies developing. In this
way competition for nutrients is eliminated, and the apparent colony count
is higher, being closer to the true number viable cells.
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