Microbiology History

Antony van Leeuwenhoek (1632-1723)

. . . my work, which I've done for a long time, was not pursued in order to gain the praise I now enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in most other men. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof. Antony van Leeuwenhoek. Letter of June 12, 1716

Antony van Leeuwenhoek was an unlikely scientist. A tradesman of Delft, Holland, he came from a family of tradesmen, had no fortune, received no higher education or university degrees, and knew no languages other than his native Dutch. This would have been enough to exclude him from the scientific community of his time completely. Yet with skill, diligence, an endless curiosity, and an open mind free of the scientific dogma of his day, Leeuwenhoek succeeded in making some of the most important discoveries in the history of biology. It was he who discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes and rotifers, and much more. His researches, which were widely circulated, opened up an entire world of microscopic life to the awareness of scientists. Leeuwenhoek was born in Delft on October 24, 1632. (His last name, incidentally, often is quite troublesome to non-Dutch speakers: "layu-wen-hook" is a passable English approximation.) His father was a basket-maker, while his mother's family were brewers. Antony was educated as a child in a school in the town of Warmond, then lived with his uncle at Benthuizen; in 1648 he was apprenticed in a linen-draper's shop. Around 1654 he returned to Delft, where he spent the rest of his life. He set himself up in business as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor city official. In 1676 he served as the trustee of the estate of the deceased and bankrupt Jan Vermeer, the famous painter, who had had been born in the same year as Leeuwenhoek and is thought to have been a friend of his. And at some time before 1668, Antony van Leeuwenhoek learned to grind lenses, made simple microscopes, and began observing with them. He seems to have been inspired to take up microscopy by having seen a copy of Robert Hooke's illustrated book Micrographia, which depicted Hooke's own observations with the microscope and was very popular.

Leeuwenhoek is known to have made over 500 "microscopes," of which fewer than ten have survived to the present day. In basic design, probably all of Leeuwenhoek's instruments -- certainly all the ones that are known -- were simply powerful magnifying glasses, not compound microscopes of the type used today. A drawing of one of Leeuwenhoek's "microscopes" is shown at the left. Compared to modern microscopes, it is an extremely simple device, using only one lens, mounted in a tiny hole in the brass plate that makes up the body of the instrument. The specimen was mounted on the sharp point that sticks up in front of the lens, and its position and focus could be adjusted by turning the two screws. The entire instrument was only 3-4 inches long, and had to be held up close to the eye; it required good lighting and great patience to use.

Compound microscopes (that is, microscopes using more than one lens) had been invented around 1595, nearly forty years before Leeuwenhoek was born. Several of Leeuwenhoek's predecessors and contemporaries, notably Robert Hooke in England and Jan Swammerdam in the Netherlands, had built compound microscopes and were making important discoveries with them. These were much more similar to the microscopes in use today. Thus, although Leeuwenhoek is sometimes called "the inventor of the microscope," he was no such thing.

However, because of various technical difficulties in building them, early compound microscopes were not practical for magnifying objects more than about twenty or thirty times natural size. Leeuwenhoek's skill at grinding lenses, together with his naturally acute eyesight and great care in adjusting the lighting where he worked, enabled him to build microscopes that magnified over 200 times, with clearer and brighter images than any of his colleagues could achieve. What further distinguished him was his curiosity to observe almost anything that could be placed under his lenses, and his care in describing what he saw. Although he himself could not draw well, he hired an illustrator to prepare drawings of the things he saw, to accompany his written descriptions. Most of his descriptions of microorganisms are instantly recognizable.

In 1673, Leeuwenhoek began writing letters to the newly-formed Royal Society of London, describing what he had seen with his microscopes -- his first letter contained some observations on the stings of bees. For the next fifty years he corresponded with the Royal Society; his letters, written in Dutch, were translated into English or Latin and printed in the Philosophical Transactions of the Royal Society, and often reprinted separately. To give some of the flavor of his discoveries, we present extracts from his observations, together with modern pictures of the organisms that Leeuwenhoek saw.

In a letter of September 7, 1674, Leeuwenhoek described observations on lake water, including an excellent description of the green charophyte alga Spirogyra: "Passing just lately over this lake, . . . and examining this water next day, I found floating therein divers earthy particles, and some green streaks, spirally wound serpent-wise, and orderly arranged, after the manner of the copper or tin worms, which distillers use to cool their liquors as they distil over. The whole circumference of each of these streaks was about the thickness of a hair of one's head. . . all consisted of very small green globules joined together: and there were very many small green globules as well."

A letter dated December 25, 1702, gives descriptions of many protists, including this ciliate, Vorticella: "In structure these little animals were fashioned like a bell, and at the round opening they made such a stir, that the particles in the water thereabout were set in motion thereby. . . And though I must have seen quite 20 of these little animals on their long tails alongside one another very gently moving, with outstretched bodies and straightened-out tails; yet in an instant, as it were, they pulled their bodies and their tails together, and no sooner had they contracted their bodies and tails, than they began to stick their tails out again very leisurely, and stayed thus some time continuing their gentle motion: which sight I found mightily diverting."

On September 17, 1683, Leeuwenhoek wrote to the Royal Society about his observations on the plaque between his own teeth, "a little white matter, which is as thick as if 'twere batter." He repeated these observations on two ladies (probably his own wife and daughter), and on two old men who had never cleaned their teeth in their lives. Looking at these samples with his microscope, Leeuwenhoek reported how in his own mouth: "I then most always saw, with great wonder, that in the said matter there were many very little living animalcules, very prettily a-moving. The biggest sort. . . had a very strong and swift motion, and shot through the water (or spittle) like a pike does through the water. The second sort. . . oft-times spun round like a top. . . and these were far more in number." In the mouth of one of the old men, Leeuwenhoek found "an unbelievably great company of living animalcules, a-swimming more nimbly than any I had ever seen up to this time. The biggest sort. . . bent their body into curves in going forwards. . . Moreover, the other animalcules were in such enormous numbers, that all the water. . . seemed to be alive." These were among the first observations on living bacteria ever recorded.

Leeuwenhoek looked at animal and plant tissues, at mineral crystals and at fossils. He was the first to see microscopic foraminifera, which he described as "little cockles. . . no bigger than a coarse sand-grain." He discovered blood cells, and was the first to see living sperm cells of animals. He discovered microscopic animals such as nematodes and rotifers. The list of his discoveries goes on and on. Leeuwenhoek soon became famous as his letters were published and translated. In 1680 he was elected a full member of the Royal Society, joining Robert Hooke, Henry Oldenburg, Robert Boyle, Christopher Wren, and other scientific luminaries of his day -- although he never attended a meeting. In 1698 he demonstrated circulation in the capillaries of an eel to Tsar Peter the Great of Russia, and he continued to receive visitors curious to see the strange things he was describing. He continued his observations until the last days of his life. After his death on August 30, 1723, the pastor of the New Church at Delft wrote to the Royal Society:

. . . Antony van Leeuwenhoek considered that what is true in natural philosophy can be most fruitfully investigated by the experimental method, supported by the evidence of the senses; for which reason, by diligence and tireless labour he made with his own hand certain most excellent lenses, with the aid of which he discovered many secrets of Nature, now famous throughout the whole philosophical World.

British scientist Brian J. Ford has rediscovered some of Leeuwenhoek's original specimens in the archives of the Royal Society of London. His study of these historic specimens and other material, using Leeuwenhoek's own microscopes and other single-lens microscopes, has shown how remarkably good a scientist and craftsman Leeuwenhoek really was. Here's the full story of Dr. Ford's research. Berkeley, California resident Al Shinn manufactures replicas of Leeuwenhoek microscopes. He has also made plans and instructions available, for those who would like to make their own Leeuwenhoek-type microscopes.

Significant Events Of The Last 125 Years

1861 Pasteur introduced the terms aerobic and anaerobic in describing the growth of yeast at the expense of sugar in the presence or absence of oxygen. He observed that more alcohol was produced in the absence of oxygen when sugar is fermented, which is now termed the Pasteur effect.

Pasteur, L. "Animalcules infusoires vivant sans gaz oxygene libre et determinant des fermentations." Compt. Rend. Acad. Sci. (Paris) 52:344-347, 1861

1865

After twenty years of freedom from the disease, Great Britain experiences an epizootic of rinderpest; in two years, 500,000 cattle die. Government inquiries into the disease and possible policy approaches elicit testimony which illustrates in some depth contemporary views regarding epidemiology and the germ theory of disease.

1. Minutes of Evidence Taken before the Cattle Plague Commissioners 11th Oct. 1865. John Simon, Esq., (Medical Officer of the Privy Council, F.R.S., and Surgeon of St. Thomas's Hospital,) examined.

2. THIRD REPORT OF THE COMMISSIONERS appointed to inquire into the ORIGIN and NATURE, &c. of the CATTLE PLAGUE; with AN APPENDIX. Presented to both Houses of Parliament by Command of Her Majesty. 1866

The Origin and Nature of the 1865 British Cattle Plague

1870 Thomas H. Huxley's Biogenesis and Abiogenesis address is the first clear statement of the basic outlines of modern Darwinian science on the question of the origin of life. The terms "biogenesis" (for life only from pre-existing life) and "abiogenesis" (for life from nonliving materials, what had previously been called spontaneous generation) as used by Huxley in this speech have become the standard terms for discussing the subject of how life originates. The speech offered powerful support for Pasteur's claim to have experimentally disproved spontaneous generation. The speech was also Huxley's attempt to define an orthodox Darwinian position on the question, and attempt to define as "non-Darwinian" all those Darwin supporters who believed that spontaneous generation up to the present day was an essential requirement of evolutionary science. Henry Charlton Bastian was the most prominent leader of that faction of Darwinians, though Huxley was so successful in defining them out of the story that very few people today even realize that there WERE Darwinians who were serious, talented evolutionary scientists, yet also thought abiogenesis was necessary in evolution up to the present day.

Biogenesis and Abiogenesis

James Strick. 1999. Darwinism and the Origin of Life: the Role of H.C. Bastian in the British Spontaneous Generation Debates, 1868-1873. Journal of the History of Biology, 32:1-42 [pdf ]

1872 Ferdinand J. Cohn contributes to the founding of the science of bacteriology. In the publication Ueber Bakterien, he discusses the role of microorganisms in the cycling of elements in nature. In 1875, Cohn will publish an early classification of bacteria, using the genus name, Bacillus, for the first time.

Cohn, F. 1872. Ueber Bakterien, die kleinsten lebenden Wesen. Lüedritz'sche Verlagsbuchhandlung Carl Habel, Berlin.

Cohn, F., 1875. Untersuchungen ueber Bakterien. Beitraege zur Biologie der Planzen 1:127-222 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p210 [pdf]

Ferdinand Cohn, a Founder of Modern Microbiology, ASM News 65. 1999.p.547

The German botanist Brefeld reported growing fungal colonies from single spores on gelatin surfaces. Prior to this innovation that resulted in the isolation of pure culture of microorganisms, pigmented bacterial colonies were isolated by the German biologist Schroeter on slices of potato incubated in a moist environment.

Brefeld, O. Botanische Untersuchungen uber Schimmelpilze, Heft I, Mucor mucedo, Chaetocladium Jones ii, Piptocephalis Fresiana: Zygomyceten, Leipzig, 1872.

Schroeter, J. "Ueber einige durch Bacterien gebildete Pigmente." Beitr. Z. Biol. D. Pflanzen 1:2, 109-126.

1876 Robert Koch publishes a paper on his work with anthrax, pointing explicitly to a bacterium as the cause of this disease. This validates the germ theory of disease. Prior, in 1872, he was approved as a district medical officer in Poland where he discovered anthrax was endemic. His work on anthrax was presented and his papers on the subject were published under the auspices of Ferdinand Cohn.

Koch, R. 1876. Untersuchungen ueber Bakterien V. Die Aetiologie der Milzbrand-Krankheit, begruendent auf die Entwicklungsgeschichte des Bacillus Anthracis. Beitr. z. Biol. D. Pflanzen 2: 277-310. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p89 [pdf ].

1877

Jean Jacques Theophile Schloesing proves that nitrification is a biological process in the soil by using chloroform vapors to inhibit the production of nitrate. One of the greatest practical applications of this knowledge was in the treatment of sewerage.

Schloesing, J. and A. Muntz. 1877. Sur la Nitrification par les Ferments Organises. Comptes Rendus de l'Academie des Sciences, Paris, LXXXIV: 301-303.

Robert Koch dries films of bacteria, stains them with methylene blue and then photographs them. He uses cover slips to prepare permanent visual records.

Koch, R. 1877. Verfahren zur Untersuchung, zum Conservieren und Photogaphiren der Bakterien. Beitraege zur Biologie der Pflanzen. 2: 399-434

John Tyndall publishes his method for fractional sterilization and clarifies the role of heat resistant factors (spores) in putrefaction. Tyndall's conclusion adds a final footnote to the work of Pasteur and others in proving that spontaneous generation is impossible.

New Details Add to Our Understanding of Spontaneous Generation Controversies, ASM News 63, 1997. p.193 [pdf]

Tyndall, J. 1877. On Heat as a Germicide when Discontinuously Applied," Proceedings of the Royal Society of London. 25:569

1878 Thomas Burrill demonstrates for the first time a bacterial disease of plants; Micrococcus amylophorous causes pear blight.

Bacteria as the Cause of Disease in Plants: A Historical Perspective, ASM News 45, 1979. p.1 [pdf]

Burrill, Thomas Jonathan. 1878. Pear blight. Trans. Ill. State Hort. Soc., 114-116.

Joseph Lister publishes his study of lactic fermentation of milk, demonstrating the specific cause of milk souring. His research is conducted using the first method developed for isolating a pure culture of a bacterium, which he names Bacterium lactis.

Lister, J. 1878. On lactic fermentation and its bearing on pathology. Trans Path. Soc., Lond. xxix: 425-67. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p58 [pdf]

Serious attention to the trypanosomes of mammals was drawn by the work of Timothy Lewis on the parasite of Indian rats (Trypanosoma lewisi), the importance of which was realized after Griffith Evans (1880) discovered the pathogenic trypanosome in horses and camels in India (Trypanosoma evansi)

Lewis, T.R. (1878) "The microscopic organisms found in the blood of man and animals and their relation to disease." Ann. Rpt. San. Commis. Govt. India, Calcutta 14:157

Lewis, T.R. (1879) "Flagellated organisms in the blood of healthy rats." Quart. J. Micr. Sci. 19:109

Evans, G. (1880) "Report on the 'surra' disease in the Dera Ismail Khan District." Punjab Govt. Milit. Dept. No. 493:446

1879 Albert Neisser identifies Neisseria gonorrhoeoe, the pathogen that causes gonorrhea. He may be the first to attribute a chronic disease to a microbe.

Neisser, A. 1879. Ueber eine der Gonorrhoe eigenthumliche Micrococcusform. Vorlaufige Mitteilung. Cbl. F. d. Med. Wiss. 28: 497-500.

1880 Louis Pasteur develops a method of attenuating a virulent pathogen, the agent of chicken cholera, so it would immunize and not cause disease. This is the conceptual break-though for establishing protection against disease by the inoculation of a weakened strain of the causative agent. Pasteur uses the word "attenuated" to mean weakened. As Pasteur acknowledged, the concept came from Jenner's success at smallpox vaccination.

Plasmids, Pasteur, and Anthrax, ASM News 49,1983. p.320 [pdf]

Pasteur, L. 1880. Sur les maladies virulentes et en particulier sur la maladie appelee vulgairement cholera des poules. Compt. Rend. Acad. Sc. 90: 239-248.

Pasteur, L. 1880. De l'attenuation du virus cholera des poules. Compte rend. Acad. se. 91: 673-680 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p126 [pdf]

C. L. Alphonse Laveran finds malarial parasites in erythrocytes of infected individuals and shows that the parasite enters the organism and replicates. Laveran was awarded the Noble Prize in Medicine or Physiology in 1907

1907 Nobel Prize

Laveran, A. 1880. A new parasite found in the blood of malarial patients. Parasitic origin of malarial attacks. Bull. mem. soc. med. hosp. Paris. 17: 158-164.

1881 Robert Koch struggles with the disadvantages of using liquid media for certain experiments. He seeks out alternatives, and first uses an aseptically cut slice of a potato as a solid culture medium. He also turns to gelatin, which is added to culture media; the resulting mixture is poured onto flat glass plates and allowed to gel. The plate technique is used to isolate pure cultures of bacteria from colonies growing on the surface of the plate. Koch publishes his Methods for the Study of Pathogenic Organisms in which he describes his success with solidified culture media.

Koch, R. 1881. Zur Untersuchung von pathogenen Organismen. Mitth. a. d. Kaiserl.

Gesundheitsampte 1: 1-48. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p101 [pdf]

Paul Ehrlich refines the use of the dye methylene blue in bacteriological staining and uses it to stain the tubercule bacillus. He shows the dye binds to the bacterium and resists decoloration with an acid alcohol wash..

Ehrlich, P. 1881 Ueber das Methylenblau und seine klinisch-bakterioskopische Verwerthung. Ztschr. f. klin. Med. ii: 710-713.

Ehrlich, P. 1882. Aus dem Verein fur innere Medicin zu Berlin. Deutsche medizinische Wochenschrift 8:269-270 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p118 [pdf]

Koch systematically investigated the efficacy of chemical disinfectants demonstrating that carbolic acid used by Lister in aseptic surgery was merely bacteriostatic and not bactericidal. He first recognized that disinfection depended on the chemical concentration and contact time. Anthrax spores were dried on silk threads, exposed to disinfectants, washed with sterile water and cultured to evaluate a range of chemicals.

1882-1891

1882

Angelina Fannie and Walther Hesse in Koch?s laboratory use agar, an extract of algae, as a solidifying agent to prepare solid media for growing microbes. Fannie suggests the use of agar-agar after leanring of it from friends who cook. Agar replaces gelatin because it remains solid at temperatures up to 100 degrees centigrade, it is clear, and it resists digestion by bacterial enzymes.

Walther and Angelina Hesse: Early Contributors to Bacteriology, ASM News 58, 1992. p.425 [pdf]

No formal article was published but see:

Hitchins, Arthur Parker and Morris C. Leikind, 1939. The Introduction of Agar-Agar Into Bacteriology. Journal of Bacteriology. 37: 485-493.

Robert Koch also mentions the cultivation of bacteria in agar-agar in The Aetiology of Tuberculosis. See below.

Robert Koch isolates the tubercule bacillus, Mycobacterium tuberculosis. The search for the tubercule bacillus is more difficult that anthrax. He finally isolates the bacillus from the tissues of a workman and stains them with methylene blue, yielding blue colored rods with bends and curves. He injects the tissues from people who had died into animals and then grows the bacilli he isolates into pure cultures.

Koch, R. 1882. Die Aetiologie der Tuberculose. Berl. Klin. Wchnschr., xix: 221-230. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p109 [pdf]

1883

Edward Theodore Klebs and Fredrich Loeffler independently discover Corynebacterium diphtheriae, which causes diphtheria. Loeffler later shows that the bacterium secretes a soluble substance that affects organs beyond sites where there is physical evidence of the organism.

Klebs, E. 1883. Ueber Diphtherie. Verh. D. Congresses f. Inn. Med., II. Congr.Bergmann Weisbaden. 139-154.

Loeffler, F. 1884. Utersuchung uber die Bedeutung der Mikroorganismen fir die Entstehung der Diptherie beim Menschen, bei der taube und beim Kalbe. Mitth. a. d. kaiserl. Gesundheitsampte. Ii: 421-499.

Ulysse Gayon and Gabriel Dupetit isolate in pure culture two strains of denitrifying bacteria. They show that individual organic compounds, such as sugars and alcohols, can replace complex organics and serve as reductants for nitrate, as well as serving as carbon sources.

1986: Centenary of the Isolation of Denitrifying Bacteria, ASM News 52, 1986. p.627 [pdf]

Gayon, U., and G. Dupetit. 1883. La fermentation des nitrates. Mem. Soc. Sci. Phys. Nat. Bordeaux Ser. 2. 5: 35-36.

1884

Ilya Ilich Metchnikoff demonstrates that certain body cells move to damaged areas of the body where they consume bacteria and other foreign particles. He calls the process phagocytosis. He proposes a theory of cellular immunity. With Ehrlich, Metchnikoff is awarded the Noble Prize in Medicine or Physiology in 1908

1908 Nobel Prize

Centennial of the Rise of Cellular Immunology: Discovery at Messina, ASM News 48, 1982. p.558 [pdf]Metschnikoff, E. 1884. Ueber eine Sprosspilzkrankheit der Daphnien. Beitrag zur Lehre uber den Kampf der Phagocyten gegen Krankheitserrenger, Archiv f. pathologische Anatomie und Physiologie und f. klinische Medicin, 96: 177-195. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p132 [pdf]

Robert Koch puts forth what will become his best-known work, a set of postulates, or standards of proof involving the tubercle bacillus. Koch's postulates are published in a work titled the The Etiology of Tuberculosis. The paper includes a demonstration of three major facts: 1) the presence of the tubercule bacillus (as proved by staining) in tubercular lesions of various organs of humans and animals, 2) the cultivation of the organisms in pure culture on blood serum, and 3) the production of tuberculosis at will by its inoculation into guinea pigs. Koch was awarded the Nobel Prize in Medicine or Physiology in 1905

1905 Nobel Prize

The Etiology of Tuberculosis: A Tribute to Robert Koch on the Occasion of the Centenary of His Discovery of the Tubercule Bacillus, ASM News 48, 1982. p.248 [pdf]

Preface to Brock's Robert Koch: A Life in Medicine and Bacteriology by James Strick [pdf]

Koch, Robert. 1884. Die Aetiologie der Tuberkulose, Mittheilungen aus dem Laiserlichen Gesundheitsampte. 2: 1-88. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p116 [pdf]

Hans Christian J. Gram develops a dye system for identifying bacteria [the Gram stain]. Bacteria which retain the violet dye are classified as gram-positive. The distinction in staining is later correlated with other biochemical and morphological differences.

Gram, C. 1884. Ueber die isolirte Farbung der Schizomyceten in Schitt-und Trockenpreparaten, Fortschritte der Medicin, 2: 185-189. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p215 [pdf]

Together with Pasteur, the French firm Chamberland's Autoclave, develops a chamber to sterilize materials using superheated steam.

Chamberland, C. 1884. Sur un filtre donnant de l'eau physiologiquement pure. Compt. Rend. Acad. d. sc. Paris. xcix: 247.

1885

As part of his rabies research, Louis Pasteur oversees injections of the child Joseph Meister with "aged" spinal cord allegedly infected with rabies virus. Pasteur uses the term "virus" meaning poison, but has no idea of the nature of the causitive organism. Although the treatment is successful, the experiment itself is an ethical violation of research standards. Pasteur knew he was giving the child successively more dangerous portions.

Pasteur: High Priest of Microbiology, ASM News 61, 1995. p.575 [pdf]

Pasteur's Dilemma: The Road Not Taken. ASM News vol. 40, 1974, p. 703 [pdf]

Preface to René Dubos' Pasteur and Modern Science by Gerald L. Geison [pdf]

Pasteur, L. 1885. Methode pour prevenir la rage apres morsure, Compt rend. Acd. Sc. 101: 765-773.

Paul Ehrlich espouses the theory that certain chemicals, such as dyes, affect bacterial cells and reasoned that these chemicals could be toxic against microbes, work that lays the foundation for his development of arsenic as a treatment for syphilis.

Ehrlich, P. 1885. Das Sauerstoff-Bedurfniss des Organismus, eine farbanalytische Studie. Hirschwald, Berlin.

Theodor Escherich identifies a bacterium, that is a natural inhabitant of the human gut, which he names Bacterium coli. He shows that certain strains are responsible for infant diarrhea and gastroenteritis.

Escherich, T. 1885. Die Darmbakterien des Neugeborenen und Sauglings, Fortschr.d. Med. 3: 515-522; 251-251,

1886

Theobald Smith and D. E. Salmon inject heated killed whole cell vaccine of hog cholera into pigeons and demonstrate immunity to subsequent administration of a live microbial culture. The organism is a bacterium and unrelated to hog cholera or swine plague disease, which is caused by a virus.

Salmon, D. E. and T. Smith. On a new method of producing immunity from contagious diseases, Proceedings of the Biological Society, (Washington, D. C.) 3: 29-33.

John Brown Buist devises a method for staining and fixing lymph matter from a cowpox vesicle. Although he believes the tiny bodies he sees are spores, he is nonetheless the first person to see (and photograph) a virus.

Buist, John Brown. "The life history of the micro-organisms associated with variola and vaccinia...." Proc. R. Soc. Edinb., 13:603-20.

1887

Sergei Winogradsky studies Beggiatoa and determines that it can use inorganic H2S as an energy source and CO2 as a carbon source. He establishes the concept of autotrophy and its relationship to natural cycles.

Winogradsky, S. 1887. Uber Schwefelbacterien. Botanische Zeitung. XLV: 489-507.

Julius Richard Petri working in Koch's laboratory, introduces a new type of culture dish for semi-solid media. The dish has an overhanging lid that keeps contaminants out.

Petri, R. J. 1887. Eine kleine Modification des Koch'schen Plattenverfahrens. Centralbl. F. Bakteriol., etc., 1: 279-280 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p218 [pdf]

1888

The Institut Pasteur is founded in France in November.

Emile Roux and Alexandre Yersin show that Cornyebacterium diphtheriae affects tissues and organs by a toxin. They use a filtrate from cells that can directly kill laboratory animals.

From "Diphtheritic" Poison to Molecular Toxinology, ASM News 53, 1987. p.547 [pdf]

Roux E. and A. Yersin. 1888. Contribution a l'etude de la diphtherie. Ann. Inst. Pasteur 2: 629-661. 3: 273.

Martinus Beijerinck uses enrichment culture, minus nitrogenous compounds, to obtain a pure culture of the root nodule bacterium Rhizobium, demonstrating that enrichment culture creates the conditions for optimal growth of a desired bacterium.

Early Biotechnology: The Delft Connection, ASM News 59, 1993. p.401 [pdf]

Beijerinck, M. 1888. Die Bakterien de Papilionaceenknollchen. Botanische Zeitung, Vol. 46: 725-804. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p220 [pdf]

Hellriegel and Wilfarth describe symbiotic nitrogen fixation by nodulated legumes. Hellriegel first reported this to a scientific meeting in September 1886, and published a somewhat more extensive paper a few weeks later. The 1888 publication with Wilfarth is considered to be "the classical paper."

Hellriegel, H, and Wilfarth, H. "Untersuchungen uber die Stickstoffnahrung der Gramineen und Leguminosen." Beilageheft zu der Zeitschrift des Vereins fur Rubenzucker-Industrie Deutschen Reichs, 234 pp., 1888

1889

A. Charrin and J. Roger discover that bacteria can be agglutinated by serum.

Charrin, A. and J. Roger. 1889. Note sur le developpement des microbes pathogens dans le serum des animaux vaccines. Soc. De Biol. 9e ser., I: 667-669.

Kitasato obtained the first pure culture of the strict anaerobic pathogen, the tetanus bacillus Clostridium tetani. Taking advantage of the fact that the spores of the organism are extremely heat-resistant, he heated a mixed culture of C. tetani and other bacteria at 80 degrees for one hour, then cultivated them in a hydrogen atmosphere.

Kitasato, S. "Ueber den Tetanusbacillus." Ztschr. Hyg. u. Infektionskrank. 7:225-234, 1889

1890

Emil von Behring and Shibasaburo Kitasato working together in Berlin in 1890 announce the discovery of diphtheria antitoxin serum, the first rational approach to therapy of infectious diseases. They inject a sublethal dose of diphtheria filtrate into animals and produce a serum that is specifically capable of neutralizing the toxin. They then inject the antitoxin serum into an uninfected animal to prevent a subsequent infection. Behring, trained as a surgeon, was a researcher for Koch. Kitasato was Koch's first student at the Institute of Hygiene. Behring was awarded the Nobel Prize in Medicine or Physiology in 1901

1901 Nobel Prize

Behring, E. 1890. Untersuchungen ueber das Zustandekommen der Diphtherie-Immunitat bei Thieren. Dt. Med. Wochenschr. 16: 1145-1148. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p141 [pdf]

Behring, E. and Kitasato, S. 1890. Ueber das Zustandekommen der Diphtherie-Immunitat und der Tetanus-Immunitat bei thieren. Deutsche medizinsche Wochenschrift 16:1113-1114 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p138 [pdf]

Sergei Winogradsky succeeds in isolating nitrifying bacteria from soil. During the period 1890-1891, Winogradsky performs the major definitive work on the organisms responsible for the process of nitrification in nature.

Winogradsky, S. 1890. Recherches sur les Organismes de la Nitrification. Computer Rendu Vol 110: 1013-1016 In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p231 [pdf]

1891

Paul Ehrlich proposes that antibodies are responsible for immunity. He shows that antibodies form against the plant toxins ricin and abrin. With Metchnikoff, Ehrlich is jointly awarded the Nobel Prize in Medicine or Physiology in 1908

1908 Nobel Prize

Christiaan Eijkman was born on August 11, 1858, at Nijkerk in Gelderland (The Netherlands), the seventh child of Christiaan Eijkman, the headmaster of a local school, and Johanna Alida Pool.

A year later, in 1859, the Eijkman family moved to Zaandam, where his father was appointed head of a newly founded school for advanced elementary education. It was here that Christiaan and his brothers received their early education. In 1875, after taking his preliminary examinations, Eijkman became a student at the Military Medical School of the University of Amsterdam, where he was trained as a medical officer for the Netherlands Indies Army, passing through all his examinations with honours.

From 1879 to 1881, he was an assistant of T. Place, Professor of Physiology, during which time he wrote his thesis On Polarization of the Nerves, which gained him his doctor's degree, with honours, on July 13, 1883. That same year he left Holland for the Indies, where he was made medical officer of health first in Semarang later at Tjilatjap, a small village on the south coast of Java, and at Padang Sidempoean in W. Sumatra. It was at Tjilatjap that he caught malaria which later so impaired his health that he, in 1885, had to return to Europe on sick-leave.

For Eijkman this was to prove a lucky event, as it enabled him to work in E. Forster's laboratory in Amsterdam, and also in Robert Koch's bacteriological laboratory in Berlin; here he came into contact with A. C. Pekelharing and C. Winkler, who were visiting the German capital before their departure to the Indies. In this way medical officer Christiaan Eijkman was seconded as assistant to the Pekelharing-Winkler mission, together with his colleague M. B. Romeny. This mission had been sent out by the Dutch Government to conduct investigations into beriberi, a disease which at that time was causing havoc in that region.

In 1887, Pekelharing and Winkler were recalled, but before their departure Pekelharing proposed to the Governor General that the laboratory which had been temporarily set up for the Commission in the Military Hospital in Batavia should be made permanent. This proposal was readily accepted, and Christiaan Eijkman was appointed its first Director, at the same time being made Director of the "Dokter Djawa School" (Javanese Medical School). Thus ended Eijkman's short military career - now he was able to devote himself entirely to science.

Eijkman was Director of the "Geneeskundig Laboratorium" (Medical Laboratory) from January 15, 1888 to March 4, 1896, and during that time he made a number of his most important researches. These dealt first of all with the physiology of people living in tropical regions. He was able to demonstrate that a number of theories had no factual basis. Firstly he proved that in the blood of Europeans living in the tropics the number of red corpuscles, the specific gravity, the serum, and the water content, undergo no change, at least when the blood is not affected by disease which will ultimately lead to anaemia. Comparing the metabolism of the European with that of the native, he found that in the tropics as well in the temperate zone, this is entirely governed by the work carried out. Neither could he find any disparity in respiratory metabolism, perspiration, and temperature regulation. Thus Eijkman put an end to a number of speculations on the acclimatization of Europeans in the tropics which had hitherto necessitated the taking of various precautions.

But Eijkman's greatest work was in an entirely different field. He discovered, after the departure of Pekelharing and Winkler, that the real cause of beriberi was the deficiency of some vital substance in the staple food of the natives, which is located in the so-called "silver skin" (pericarpium) of the rice. This discovery has led to the concept of vitamins. This important achievement earned him the Nobel Prize in Physiology or Medicine for 1929. This late recognition of his outstanding merits has ended all criticism of his work. In addition to his work on beriberi, he occupied himself with other problems such as arach fermentation, and indeed still had time to write two textbooks for his students at the Java Medical School, one on physiology and the other on organic chemistry.

In 1898 he became successor to G. Van Overbeek de Meyer, as Professor in Hygiene and Forensic Medicine at Utrecht. His inaugural speech was entitled Over Gezondheid en Ziekten in Tropische Gewesten (On health and diseases in tropical regions). At Utrecht, Eijkman turned to the study of bacteriology, and carried out his well-known fermentation test, by means of which it can be readily established if water has been polluted by human and animal defaecation containing coli bacilli. Another research was into the rate of mortality of bacteria as a result of various external factors, whereby he was able to show that this process could not be represented by a logarithmic curve. This was followed by his investigation of the phenomenon that the rate of growth of bacteria on solid substratum often decreases, finally coming to a halt. Beyerinck's auxanographic method was applied on several occasions by Eijkman, as for example during the secretion of enzymes which break down casein or bring about haemolysis, whereby he could demonstrate the hydrolysis of fats under the influence of lipases.

As a lecturer he was known for his clarity of speech and demonstration, his great practical knowledge standing him in good stead. He had a preeminently critical mind and he continuously warned his students against the acceptance of dogmas. But Eijkman did not confine himself to the University he also engaged himself in problems of water supply, housing, school hygiene, physical education; as a member of the Gezondheidsraad (Health Council) and the Gezondheidscommissie (Health Commission) he participated in the struggle against alcoholism and tuberculosis. He was the founder of the Vereeniging tot Bestrijding van de Tuberculose (Society for the struggle against tuberculosis ).

His unassuming personality has contributed to the fact that his great merits were at first not really appreciated in his own country; but anyone who had the privilege of coming into close contact with him, quickly perceived his keen intellect and extensive knowledge.

In 1907, Eijkman was appointed Member of the Royal Academy of Sciences (The Netherlands), after having been Correspondent since 1895. The Dutch Government conferred upon him several orders of knighthood, whereas on the occasion of the 25th anniversary of his professorship a fund has been established to enable the awarding of the Eijiman Medal. But the crown of all his work was the award of the Nobel Prize in 1929.

Eijkman was holder of the John Scott Medal, Philadelphia, and Foreign Associate of the National Academy of Sciences in Washington. He was also Honorary Fellow of the Royal Sanitary Institute in London.

In 1883, before his departure to the Indies, Eijkman married Aaltje Wigeri van Edema, who died in 1886. In Batavia, Professor Eijkman married Bertha Julie Louise van der Kemp in 1888; a son, Pieter Hendrik, who became a physician, was born in 1890.

He died in Utrecht, on November 5, 1930, after a protracted illness.

Paul Ehrlich was born on March 14, 1854 at Strehlen, in Upper Silesia*, Germany. He was the son of Ismar Ehrlich and his wife Rosa Weigert, whose nephew was the great bacteriologist Karl Weigert.

Ehrlich was educated at the Gymnasium at Breslau and subsequently at the Universities of Breslau, Strassburg, Freiburg-im-Breisgau and Leipzig. In 1878 he obtained his doctorate of medicine by means of a dissertation on the theory and practice of staining animal tissues. This work was one of the results of his great interest in the aniline dyes discovered by W. H. Perkin in 1853.

In 1878 he was appointed assistant to Professor Frerichs at the Berlin Medical Clinic, who gave him every facility to continue his work with these dyes and the staining of tissues with them. Ehrlich showed that all the dyes used could be classified as being basic, acid or neutral and his work on the staining of granules in blood cells laid the foundations of future work on haematology and the staining of tissues.

In 1882 Ehrlich published his method of staining the tubercle bacillus that Koch had discovered and this method was the basis of the subsequent modifications introduced by Ziehl and Neelson, which are still used today. From it was also derived the Gram method of staining bacteria so much used by modern bacteriologists.

In 1882 Ehrlich became Titular Professor and in 1887 he qualified, as a result of his thesis Das Sauerstoffbedürfnis des Organismus (The need of the organism for oxygen) as a Privatdozent (unpaid lecturer or instructor) in the Faculty of Medicine in the University of Berlin. Later he became an Associate Professor there and Senior House Physician to the Charité Hospital in Berlin.

In 1890 Robert Koch, Director of the newly established Institute for Infectious Diseases, appointed Ehrlich as one of his assistants and Ehrlich then began the immunological studies with which his name will always be associated.

At the end of 1896 an Institute for the control of therapeutic sera was established at Steglitz in Berlin and Ehrlich was appointed its Director. Here he did further important work on immunology, especially on haemolysins. He also showed that the toxin-antitoxin reaction is, as chemical reactions are, accelerated by heat and retarded by cold and that the content of antitoxin in antitoxic sera varied so much for various reasons that it was necessary to establish a standard by which their antitoxin content could be exactly measured. This he accomplished with von Behring's antidiphtheritic serum and thus made it possible to standardize this serum in units related to a fixed and invariable standard. The methods of doing this that Ehrlich then established formed the basis of all future standardization of sera. This work and his other immunological studies led Ehrlich to formulate his famous side-chain theory of immunity.

In 1897 Ehrlich was appointed Public Health Officer at Frankfurt-am-Main and when, in 1899, the Royal Institute of Experimental Therapy was established at Frankfurt, Ehrlich became its Director. He also became Director of the Georg Speyerhaus, which was founded by Frau Franziska Speyer and was built next-door to Ehrlich's Institute. These appointments marked the beginning of the third phase of Ehrlich's many and varied researches. He now devoted himself to chemotherapy, basing his work on the idea, which had been implicit in his doctorate thesis written when he was a young man, that the chemical constitution of drugs used must be studied in relation to their mode of action and their affinity for the cells of the organisms against which they were directed. His aim was, as he put it, to find chemical substances which have special affinities for pathogenic organisms, to which they would go, as antitoxins go to the toxins to which they are specifically related, and would be, as Ehrlich expressed it, «magic bullets» which would go straight to the organisms at which they were aimed.

To achieve this, Ehrlich tested, with the help of his assistants, hundreds of chemical substances selected from the even larger number of these that he had collected. He studied, among other subjects, the treatment of trypanosomiasis and other protozoal diseases and produced trypan red, which was, as his Japanese assistant Shiga showed, effective against trypanosomes. He also established, with A. Bertheim, the correct structural formula of atoxyl, the efficiency of which against certain experimental trypanosomiases was known. This work opened a way of obtaining numerous new organic compounds with trivalent arsenic which Ehrlich tested.

At this time, the spirochaete that causes syphilis was discovered by Schaudinn and Hoffmann in Berlin, and Ehrlich decided to seek a drug that would be effective especially against this spirochaete. Among the arsenical drugs already tested for other purposes was one, the 606th of the series tested, which had been set aside in 1907 as being ineffective. But when Ehrlich's former colleague Kitasato sent a pupil of his, named Hata, to work at Ehrlich's Institute, Ehrlich, learning that Hata had succeeded in infecting rabbits with syphilis, asked him to test this discarded drug on these rabbits. Hata did so and found that it was very effective.

When hundreds of experiments had repeatedly proved its efficacy against syphilis, Ehrlich announced it under the name «Salvarsan». Subsequently, further work on this subject was done and eventually it turned out that the 914th arsenical substance to which the name «Neosalvarsan» was given, was, although its curative effect was less, more easily manufactured and, being more soluble, became more easily administered. Ehrlich had, like so many other discoverers before him, to battle with much opposition before Salvarsan or Neosalvarsan were accepted for the treatment of human syphilis; but ultimately the practical experience prevailed and Ehrlich became famous as one of the main founders of chemotherapy.

During the later years of his life, Ehrlich was concerned with experimental work on tumours and on his view that sarcoma may develop from carcinoma, also on his theory of athreptic immunity to cancer.

The indefatigable industry shown by Ehrlich throughout his life, his kindness and modesty, his lifelong habit of eating little and smoking incessantly 25 strong cigars a day, a box of which he frequently carried under one arm, his invariable insistence on the repeated proof by many experiments of the results he published, and the veneration and devotion shown to him by all his assistants have been vividly described by his former secretary, Martha Marquardt, whose biography of him has given us a detailed picture of his life in Frankfurt. In Frankfurt the street in which his Institute was situated was named Paul Ehrlichstrasse after him, but later, when the Jewish persecution began, this name was removed because Ehrlich was a Jew. After the Second World War, however, whe n his birth-place, Strehlen, came under the jurisdiction of the Polish authorities, they renamed it Ehrlichstadt, in honour of its great son.

Ehrlich was an ordinary, foreign, corresponding or honorary member of no less than 81 academies and other learned bodies in Austria, Belgium, Brazil, Denmark, Egypt, Finland, France, Germany, Great Britain, Greece, Hungary, ltaly, The Netherlands, Norway, Roumania, Russia, Serbia, Sweden, Turkey, the U.S.A. and Venezuela. He held honorary doctorates of the Universities of Chicago, Göttingen, Oxford, Athens and Breslau, and was also honoured by Orders in Germany, Russia, Japan, Spain, Roumania, Serbia, Venezuela, Denmark (Commander Cross of the Danebrog Order), and Norway (Commander Cross of the Royal St. Olaf Order).

In 1887 he received the Tiedemann Prize of the Senckenberg Naturforschende Gesellschaft at Frankfurt/Main, in 1906 the Prize of Honour at the XVth International Congress of Medicine at Lisbon, in 1911 the Liebig Medal of the German Chemical Society, and in 1914 the Cameron Prize of Edinburgh. In 1908 he shared with Metchnikoff the highest scientific distinction, the Nobel Prize.

The Prussian Government elected him Privy Medical Counsel in 1897, promoted him to a higher rank of this Counsel in 1907 and, in 1911, raised him to the highest rank, Real Privy Counsel with the title of Excellency.

Ehrlich married, in 1883, Hedwig Pinkus, who was then aged 19. They had two daughters, Stephanie (Mrs. Ernst Schwerin) and Marianne (Mrs. Edmund Landau).

When the First World War broke out in 1914 he was much distressed by it and at Christmas of that year he had a slight stroke. He recovered quickly from this, but his health which had never, apart from a tuberculous infection in early life which had made it necessary for him to spend two years in Egypt, failed him, now began to decline and when, in 1915, he went to Bad Homburg for a holiday, he had, on August 20 of that year, a second stroke which ended his life.

Robert Koch was born on December 11, 1843, at Clausthal in the Upper Harz Mountains. The son of a mining engineer, he astounded his parents at the age of five by telling them that he had, with the aid of the newspapers, taught himself to read, a feat which foreshadowed the intelligence and methodical persistence which were to be so characteristic of him in later life. He attended the local high school («Gymnasium») and there showed an interest in biology and, like his father, a strong urge to travel.

In 1862 Koch went to the University of Göttingen to study medicine. Here the Professor of Anatomy was Jacob Henle and Koch was, no doubt, influenced by Henle's view, published in 1840, that infectious diseases were caused by living, parasitic organisms. After taking his M.D. degree in 1866, Koch went to Berlin for six months of chemical study and there came under the influence of Virchow. In 1867 he settled, after a period as Assistant in the General Hospital at Hamburg, in general practice, first at Langenhagen and soon after, in 1869, at Rackwitz, in the Province of Posen. Here he passed his District Medical Officer's Examination. In 1870 he volunteered for service in the Franco-Prussian war and from 1872 to 1880 he was District Medical Officer for Wollstein. It was here that he carried out the epoch-making researches which placed him at one step in the front rank of scientific workers.

Anthrax was, at that time, prevalent among the farm animals in the Wollstein district and Koch, although he had no scientific equipment and was cut off entirely from libraries and contact with other scientific workers, embarked, in spite of the demands made on him by his busy practice, on a study of this disease. His laboratory was the 4-roomed flat that was his home, and his equipment, apart from the microscope given to him by his wife, he provided for himself. Earlier the anthrax bacillus had been discovered by Pollender, Rayer and Davaine, and Koch set himself to prove scientifically that this bacillus is, in fact, the cause of the disease. He inoculated mice, by means of home-made slivers of wood, with anthrax bacilli taken from the spleens of farm animals that had died of anthrax, and found that these mice were all killed by the bacilli, whereas mice inoculated at the same time with blood from the spleens of healthy animals did not suffer from the disease. This confirmed the work of others who had shown that the disease can be transmitted by means of the blood of animals suffering from anthrax.

But this did not satisfy Koch. He also wanted to know whether anthrax bacilli that had never been in contact with any kind of animal could cause the disease. To solve this problem he obtained pure cultures of the bacilli by growing them on the aqueous humour of the ox's eye. By studying, drawing and photographing these cultures, Koch recorded the multiplication of the bacilli and noted that, when conditions are unfavourable to them, they produce inside themselves rounded spores which can resist adverse conditions, especially lack of oxygen and that, when suitable conditions of life are restored, the spores give rise to bacilli again. Koch grew the bacilli for several generations in these pure cultures and showed that, although they had had no contact with any kind of animal, they could still cause anthrax.

The results of this painstaking work were demonstrated by Koch to Ferdinand Cohn, Professor of Botany at the University of Breslau, who called a meeting of his colleagues to witness this demonstration, among whom was Professor Cohnheim, Professor of Pathological Anatomy. Both Cohn and Cohnheim were deeply impressed by Koch's work and when Cohn, in 1876, published Koch's work in the botanical journal of which he was the editor, Koch immediately became famous. He continued, nevertheless, to work at Wollstein for a further four years and during this period he improved his methods of fixing, staining and photographing bacteria and did further important work on the study of diseases caused by bacterial infections of wounds, publishing his results in 1878. In this work he provided, as he had done with anthrax, a practical and scientific basis for the control of these infections.

Koch was still, however, without adequate quarters or conditions for his work and it was not until 1880, when he was appointed a member of the «Reichs-Gesundheitsamt» (Imperial Health Bureau) in Berlin, that he was provided, first with a narrow, inadequate room, and later with a better laboratory, in which he could work with Loeffler, Gaffky and others, as his assistants. Here Koch continued to refine the bacteriological methods he had used in Wollstein. He invented new methods - «Reinkulturen» - of cultivating pure cultures of bacteria on solid media such as potato, and on agar kept in the special kind of flat dish invented by his colleague Petri, which is still in common use. He also developed new methods of staining bacteria which made them more easily visible and helped to identify them. The result of all this work was the introduction of methods by which pathogenic bacteria could be simply and easily obtained in pure culture, free from other organisms and by which they could be detected and identified. Koch also laid down the conditions, known as Koch's postulates, which must be satisfied before it can be accepted that particular bacteria cause particular diseases.

Some two years after his arrival in Berlin Koch discovered the tubercle bacillus and also a method of growing it in pure culture. In 1882 he published his classical work on this bacillus. He was still busy with work on tuberculosis when he was sent, in 1883, to Egypt as Leader of the German Cholera Commission, to investigate an outbreak of cholera in that country. Here he discovered the vibrio that causes cholera and brought back pure cultures of it to Germany. He also studied cholera in India.

On the basis of his knowledge of the biology and mode of distribution of the cholera vibrio, Koch formulated rules for the control of epidemics of cholera which were approved by the Great Powers in Dresden in 1893 and formed the basis of the methods of control which are still used today. His work on cholera, for which a Prize of 100,000 German Marks was awarded to him, also had an important influence on plans for the conservation of water supplies.

In 1885 Koch was appointed Professor of Hygiene in the University of Berlin and Director of the newly established Institute of Hygiene in the University there. In 1890 he was appointed Brigadier General (Generalarzt) Class I and Freeman of the City of Berlin. In 1891 he became an Honorary Professor of the Medical Faculty of Berlin and Director of the new Institute for Infectious Diseases, where he was fortunate to have among his colleagues, such men as Ehrlich, von Behring and Kitasato, who themselves made great discoveries.

During this period Koch returned to his work on tuberculosis. He sought to arrest the disease by means of a preparation, which he called tuberculin, made from cultures of tubercle bacilli. He made two preparations of this kind called the old and the new tuberculin respectively, and his first communication on the old tuberculin aroused considerable controversy. Unfortunately, the healing power that Koch claimed for this preparation was greatly exaggerated and, because hopes raised by it were not fulfilled, opinion went against it and against Koch. The new tuberculin was announced by Koch in 1896 and the curative value of this also was disappointing; but it led, nevertheless, to the discovery of substances of diagnostic value. While this work on tuberculin was going on, his colleagues at the Institute for Infectious Diseases, von Behring, Ehrlich and Kitasato, carried out and published their epoch-making work on the immunology of diphtheria (see the biographies of Ehrlich and von Behring).

In 1896 Koch went to South Africa to study the origin of rinderpest and although he did not identify the cause of this disease, he succeeded in limiting the outbreak of it by injection into healthy farm-stock of bile taken from the gall bladders of infected animals. Then followed work in India and Africa on malaria, blackwater fever, surra of cattle and horses and plague, and the publication of his observations on these diseases in 1898. Soon after his return to Germany he was sent to Italy and the tropics where he confirmed the work of Sir Ronald Ross in malaria and did useful work on the aetiology of the different forms of malaria and their control with quinine.

It was during these later years of his life that Koch came to the conclusion that the bacilli that caused human and bovine tuberculosis are not identical and his statement of this view at the International Medical Congress on Tuberculosis in London in 1901 caused much controversy and opposition; but it is now known that Koch's view was the right one. His work on typhus led to the idea, then a new one, that this disease is transmitted much more often from man to man than from drinking water and this led to new control measures.

In December, 1904, Koch was sent to German East Africa to study East Coast fever of cattle and he made important observations, not only on this disease, but also on pathogenic species of Babesia and Trypanosoma and on tickborne spirochaetosis, continuing his work on these organisms when he returned home.

Koch was the recipient of many prizes and medals, honorary doctorates of the Universities of Heidelberg and Bologna, honorary citizenships of Berlin, Wollstein and his native Clausthal, and honorary memberships of learned societies and academies in Berlin, Vienna, Posen, Perugia, Naples and New York. He was awarded the German Order of the Crown, the Grand Cross of the German Order of the Red Eagle (the first time this high distinction was awarded a medical man), and Orders from Russia and Turkey. Long after his death, he was posthumously honoured by memorials and in other ways in several countries.

In 1905 he was awarded the Nobel Prize for Physiology or Medicine. In 1906, he returned to Central Africa to work on the control of human trypanosomiasis, and there he reported that atoxyl is as effective against this disease as quinine is against malaria. Thereafter Koch continued his experimental work on bacteriology and serology.

In 1866 Koch married Emmy Fraats. She bore him his only child, Gertrud (b. 1865), who became the wife of Dr. E. Pfuhl. In 1893 Koch married Hedwig Freiberg.

Dr. Koch died on May 27, 1910, in Baden-Baden.

Sir Alexander Fleming was born at Lochfield near Darvel in Ayrshire, Scotland on August 6th, 1881. He attended Louden Moor School, Darvel School, and Kilmarnock Academy before moving to London where he attended the Polytechnic. He spent four years in a shipping office before entering St. Mary's Medical School, London University. He qualified with distinction in 1906 and began research at St. Mary's under Sir Almroth Wright, a pioneer in vaccine therapy. He gained M.B., B.S., (London), with Gold Medal in 1908, and became a lecturer at St. Mary's until 1914. He served throughout World War I as a captain in the Army Medical Corps, being mentioned in dispatches, and in 1918 he returned to St.Mary's. He was elected Professor of the School in 1928 and Emeritus Professor of Bacteriology, University of London in 1948. He was elected Fellow of the Royal Society in 1943 and knighted in 1944.

Early in his medical life, Fleming became interested in the natural bacterial action of the blood and in antiseptics. He was able to continue his studies throughout his military career and on demobilization he settled to work on antibacterial substances which would not be toxic to animal tissues. In 1921, he discovered in «tissues and secretions» an important bacteriolytic substance which he named Lysozyme. About this time, he devised sensitivity titration methods and assays in human blood and other body fluids, which he subsequently used for the titration of penicillin. In 1928, while working on influenza virus, he observed that mould had developed accidently on a staphylococcus culture plate and that the mould had created a bacteria-free circle around itself. He was inspired to further experiment and he found that a mould culture prevented growth of staphylococci, even when diluted 800 times. He named the active substance penicillin.

Sir Alexander wrote numerous papers on bacteriology, immunology and chemotherapy, including original descriptions of lysozyme and penicillin. They have been published in medical and scientific journals.

Fleming, a Fellow of the Royal College of Surgeons (England), 1909, and a Fellow of the Royal College of Physicians (London), 1944, has gained many awards. They include Hunterian Professor (1919), Arris and Gale Lecturer (1929) and Honorary Gold Medal (1946) of the Royal College of Surgeons; Williams Julius Mickle Fellowship, University of London (1942); Charles Mickle Fellowship, University of Toronto (1944); John Scott Medal, City Guild of Philadelphia (1944); Cameron Prize, University of Edinburgh (1945); Moxon Medal, Royal College of Physicians (1945); Cutter Lecturer, Harvard University (1945); Albert Gold Medal, Royal Society of Arts (1946); Gold Medal, Royal Society of Medicine (1947); Medal for Merit, U.S.A. (1947); and the Grand Cross of Alphonse X the Wise, Spain (1948).

He served as President of the Society for General Microbiology, he was a Member of the Pontifical Academy of Science and Honorary Member of almost all the medical and scientific societies of the world. He was Rector of Edinburgh University during 1951-1954, Freeman of many boroughs and cities and Honorary Chief Doy-gei-tau of the Kiowa tribe. He was also awarded doctorate, honoris causa, degrees of almost thirty European and American Universities.

In 1915, Fleming married Sarah Marion McElroy of Killala, Ireland, who died in 1949. Their son is a general medical practitioner.

Fleming married again in 1953, his bride was Dr. Amalia Koutsouri-Voureka, a Greek colleague at St. Mary's.

In his younger days he was a keen member of the Territorial Army and he served from 1900 to 1914 as a private in the London Scottish Regiment.

Dr Fleming died on March 11th in 1955 and is buried in St. Paul's Cathedral.

Sir Howard Walter Florey was born on September 24, 1898, at Adelaide, South Australia, the son of Joseph and Bertha Mary Florey. His early education was at St. Peter's Collegiate School, Adelaide, following which he went on to Adelaide University where he graduated M.B., B.S. in 1921. He was awarded a Rhodes Scholarship to Magdalen College, Oxford, leading to the degrees of B.Sc. and M.A. (1924). He then went to Cambridge as a John Lucas Walker Student. In 1925 he visited the United States on a Rockefeller Travelling Fellowship for a year, returning in 1926 to a Fellowship at Gonville and Caius College, Cambridge, receiving here his Ph.D. in 1927. He also held at this time the Freedom Research Fellowship at the London Hospital. In 1927 he was appointed Huddersfield Lecturer in Special Pathology at Cambridge. In 1931 he succeeded to the Joseph Hunter Chair of Pathology at the University of Sheffield.

Leaving Sheffield in 1935 he became Professor of Pathology and a Fellow of Lincoln College, Oxford. He was made an Honorary Fellow of Gonville and Caius College, Cambridge in 1946 and an Honorary Fellow of Magdalen College, Oxford in 1952. In 1962 he was made Provost of The Queen's College, Oxford.

During World War II he was appointed Honorary Consultant in Pathology to the Army and in 1944 he became Nuffield Visiting Professor to Australia and New Zealand.

His best-known work dates from his collaboration with Chain, which began in 1938 when they conducted a systematic investigation of the properties of naturally occurring antibacterial substances. Lysozyme, an antibacterial substance found in saliva and human tears, was their original interest, but their interest moved to substances now known as antibiotics. The work on penicillin was a result of this interest.

Penicillin had been discovered by Fleming in 1928 as a result of observations on a mould which developed on some germ culture plates but the active substance was not isolated. In 1939, Florey and Chain headed a team of British scientists, financed by a grant from the Rockefeller Foundation, whose efforts led to the successful small-scale manufacture of the drug from the liquid broth in which it grows. In 1940 a report was issued describing how penicillin had been found to be a chemotherapeutic agent capable of killing sensitive germs in the living body. Thereafter great efforts were made, with government assistance, to enable sufficient quantities of the drug to be made for use in World War II to treat war wounds.

Florey was a contributor to, and Editor of, Antibiotics (1949). He was also part-author of a book of lectures on general pathology and has had many papers published on physiology and pathology.

Dr. Florey has had many honours bestowed upon him. Among these may be mentioned the Lister Medal of the Royal College of Surgeons, the Berzelius Medal of the Swedish Medical Society, the Royal and Copley Medals of the Royal Society, the Medal of Merit of the U. S. Army, and many others.

He is President of the Royal Society since 1960 and a Fellow of the Royal College of Physicians, and among other honorary fellowships he holds is that of the Royal Australian College of Physicians.

He has been awarded honorary degrees by seventeen universities and is a member or honorary member of many learned societies and academies in the field of medicine and biology.

In 1944 he was created a Knight Bachelor.

He married Mary Ethel Hayter Reed in 1926. They have two children, Paquita Mary Joanna and Charles du Vé.

A Basic History of Plasmid Research

Plasmid Early History Time-Line:

1903: Walter S. Sutton and Theodor Boveri independently develop the hypothesis that the units of heredity are physically located on chromosomes, thus giving a physical location for heredity. 1910: Thomas Hunt Morgan describes association of heritable properties in Drosophila with a specific chromosome and begins the analysis of genes in the nucleus. 1920s-1940: Embryological observations suggest that there are hereditary determinants in the cytoplasm. 1946: Joshua Lederberg and Edward Tatum report strong evidence for a sexual phase in E. coli K-12. 1949-1951: J. Lederberg and Cavalli and Heslot find that most strains of E. coli will not mate with K-12. 1950: Andre Lwoff and Antoinette Gutmann clarify the nature of phage lysogeny. 1951: Esther Lederberg discovers the lyosgenic bacteriophage lambda in E. coli K-12. 1950s: Respiratory deficient mutants in yeast (petites) are studied by P. Slonimski and B. Ephrussi and are attributed to cytoplasmic hereditary units in the mitochondria. Mutations in Chlamydomonas are attributed to hereditary units in the chloroplasts by R. Sager. 1950-1952: William Hayes suggests that mating in E. coli is an asymmetric (unidirectional) process rather than one analogous to cell fusion and zygote formation in higher organisms. 1952: J. Lederberg reviews the literature on cell heredity and suggests the term"Plasmid" for all extrachromosomal hereditary determinants. 1952-1953: Hayes, and J. Lederberg, Cavalli, and E. Lederberg report that the ability to mate is controlled by a factor (F) that seems to be an infectious particle not associated with the chromosome. 1954: Pierre Fredéricq and colleagues show that colicines behave as genetic factors independent of the chromosome. 1958: François Jacob and Elie Wollman propose the term "Episome" to describe genetic elements such as F, colicine, and phage lambda which can exist both in association with the chromosome and independent of it. 1959: Jacob and Edward Adelberg find that the F-factor can associate with cell genes and identify F-prime factors. 1959: Alfred Kleinschmidt and R. Zahn show that DNA molecules can be studied in the EM by spreading the DNA in protein films on the surface of water. 1960-1961: T. Akiba, T. Koyama, Y. Isshiki, S. Kimua, and T. Fukushima, and T. Watanabe and T. Fukusawa describe multiple drug resistance transferred by an episome designated the R-factor. 1961: Physical experiments involving DNA labeling (either by density [Marmur et al] or radioactivity [Silver and Ozeki]) show that mating in bacteria is accompanied by transfer of DNA from the donor to the recipient. 1962: In a review on episomes, Allan Campbell proposes the reciprocal recombination of circular episome DNA molecules with the chromosomal DNA as a way to physically insert the episome DNA linearly into the chromosome. 1962: Circular DNA is found to actually exist by Walter Fiers and Robert Sinsheimer in the genome of the small phage phi-X174. 1963: Alfred Hershey shows that bacteriophage lambda can form circles in vitro by virtue of its "cohesive ends". Other circular DNAs are also reported: the E. coli genome by John Cairns, and polyoma virus DNA by Renato Dulbecco and Margerite Vogt, and by Roger Weil and Jerome Vinograd. 1967: R. Radloff, William Bauer, and J. Vinograd describe the dye-bouyant density method to separate closed circular DNA from open circles and linear DNA, thus facilitating the physical study of plasmids. 1969: M. Bazarle and D. R. Helinski show that several colicine factors are homogeneous circular DNA molecules.

Particulate Heredity The early history of the concept of a plasmid is rooted in the concept of particulate determinants of inheritance. In the first decade of the 20th century, the chromosome theory was developed and two key papers are usually cited: Johannsen and Boveri. These workers argued from diverse observations that the cytologically observable structures in the cell nucleus are the physical units that determine the Mendelian characters. Of course, it was very unclear just what a "Mendelian character" was. In the second decade of this century, Thomas Hunt Morgan and his group, in experiments with fruit flies (Drosophila melanogaster) presented evidence for the formal agreement of the behavior of several "Mendelian factors" and the behavior of the physical structures known as chromosomes. Morgan and his school generalized these results into a broad "Theory of the Gene" which held that the Mendelian factors (genes) were arranged linearly on the visible structures (chromosomes) that resided in the nucleus of every cell and which were duplicated and partitioned equally to the daughter cells during cell division. Thus was solved (at one level, at least) the age-old problem of "how like begets like". Many biologists took up this approach and gathered much evidence to supports its validity and universality.

Cytoplasmic Contributions At the same time, other biologists, working on problems of embryology and morphology, saw genes as determinants of the way an organism developed from a fertilized ovum into a mature adult. For them, genetics was not about transmission of characters across the generations, but about how gene action worked to make the organism a nearly exact copy of its parents, that is, a different version of the age-old problem of "how like begets like." For many of these biologists, the determinants of the characters involved in development and differentiation seemed to be neither obviously nuclear, nor chromosomal. Some of these genes seems to be passed on through cytoplasmic transfer. For example, in 1937 the eminent biologist Ross Harrison wrote (Science 85:372) "The prestige of success enjoyed by the gene theory might easily become a hindrance to the understanding of development by directing our attention solely to the genome, whereas cell movements, differentiation and in fact all developmental processes are actually effected by the cytoplasm. Already we have theories that refer the process of development to genic action and regard the whole performance as no more that the realization of the potencies of the genes. Such theories are altogether too one-sided."

By the mid 1930s, these cytoplasmic determinants came to be known as "plasmagenes." Plasmagenes, however, were often invoked to explain the possible mechanisms of "inheritance of acquired traits" and played directly into the schemes of the Michurinist/Lysenkoist genetics in the Soviet Union. At the time, then, plasmagenes acquired the extra baggage of Cold War ideology (See Chapter 6: The Cold War in Genetics: Jan Sapp: Beyond the Gene, Oxford, 1987).

Genes in Bacteria The existence of genes in bacteria was much debated in the first half of the 20th century. Without a visible nucleus, without visible chromosomes, without a known dimorphic sexual phase, and without many distinguishing charcteristics, it was easy to believe that bacteria were altogether different from organisms which reproduced sexually. In 1942 (Evolution: The Modern Synthesis, p. 131-132) the famous British biologist Julian Huxley wrote: "Bacteria (and a fortiori viruses, if they can be considered to be true organisms), in spite of occasional reports of a sexual cycle, appear to be not only wholly asexual but pre-mitotic. Their hereditary constitution is not differentiated into special parts with different functions. They have no genes in the sense of accurately quantized portions of hereditary substance; and therefore they have no need for the accurate division of the genetic system which is accomplished by mitosis.... We must, in fact, expect that the process of variation, heredity, and evolution in bacteria are quite different from the corresponding processes in multicellular organisms. But their secret has not yet been unraveled."

By 1946, however, the experiments of J. Lederberg and E.L. Tatum (Nature 158:558) challenged and clarified the understanding of genes in bacteria. Without dealing with the physical nature of the genetic structures in bacteria (there was considerable debate about the existence of a bacterial nucleus), Lederberg and Tatum obtained clear support for a mating system in a bacterium ( Escherichia coli, strain K-12) and in 1947 Lederberg employed Morgan's paradigm of genetic linkage, established a genetic map in E. coli based on the frequency of recombination of genetic determinants observed in standardized "matings" (Genetics 32:505). At this time, the dominant model was based on the sexual processes in higher cells: cell fusion with zygote formation, recombination, and marker segregation and cell division.

In 1949 L.L. Cavalli and H. Heslot (Nature164:1057), and in 1951, J. Lederberg (Science114:68) surveyed other strains of E. coli for their ability to mate with Lederberg's strain K-12, and found that only 9 of 140 isolates could mate with K-12. Thus, there seemed to be something peculiar about mating in E. coli. In London, William Hayes started to study the kinetics of the mating process in 1950 and at the suggestion of D. Mitchison, conceived of bacterial mating as an asymmetric process involving a gene donor and a gene acceptor. This model for bacterial mating fitted the data Hayes was obtaining in various bacterial matings much better than a classical cell fusion model, and in 1952 he published this work (Nature169:118) and presented it at meetings in the summer of 1952: James D. Watson described the event (The Double Helix. Atheneum,1968, pp. 141-142):

"Bill's appearance was the sleeper of the three day gathering; before his talk no one except Cavalli-Sforza knew he existed. As soon as he had finished his unassuming report, however, everyone in the audience knew that a bombshell had exploded in the world of Joshua Lederberg!"

The F-factor The directionality and polarity of the bacterial mating process, first suggested by Hayes, greatly clarified the understanding of bacterial genetics as studied by mating experiments. The problem of sexual compatibility, however, remained. The rather rare property of a given E. coli strain to mate was a puzzle. In 1952 J. Lederberg, L.L. Cavalli, and E.M. Lederberg (Genetics 37:720-730) and in 1953 Hayes (J. Gen. Microbiol. 8:72-88) independently reported that the ability to act as a donor in a bacterial mating was a property controlled by an "factor" designated "F" (fertility) that seemed to behave as "an infectious particle."

Lambda Bacteriophage and Colicines In the mid-1950s two other anomalous hereditary "factors" were discovered to behave as "infectious particles" as well. One was the bacteriophage lambda, a lysogenic phage found in E. coli K-12 by Esther Lederberg, and the other was the factor determining the production of colicine, a killer substance, produced by some strains of E. coli and studied by Pierre Frédéricq (originally discovered by André Gratia).

Plasmids and Episomes In a broad review of "Cell Genetics and Hereditary Symbiosis" (Physiol. Rev. 1952, pp. 403-430) Joshua Lederberg proposed that all "extrachromosomal hereditary determinants" be subsumed under the designation "plasmid." [To view excerpt from this reference Click Here] He did not distinguish nuclear or cytoplasmic location, nor the possibility of association of such determinants with the chromosome on some occasions. In a more limited review of bacterial genetic systems, F. Jacob and E. Wollman in 1958 ( Compt rend. acad. sci. 247:154) suggested that genetic elements which were optionally associated with the chromosomes of the cell be termed "episomes." [To view an excerpt from this reference Click Here]. They used the F-factor, the colicinogenic factor, and bacteriophage lambda as prototypic episomes. By this time it was known that the F- factor could become associated with the bacterial chromosome and result in the transfer of chromosomal genes with high frequency in mating experiments (Hfr strains). By the end of the 1950s the recognition of genetic determinants which were not able to be located on the genetic map in standard crosses established the concept of "plasmid" (episome was used rather interchangeably with plasmid by some, but William Hayes, for one, calling the F-factor "a small, supernumerary chromosome," stated (The Genetics of Bacteria and their Viruses, 2nd ed. Blackwell, 1964, pp. 747-748):

"We think the word 'episome,' although an excellent substitute for 'plasmid,' has become a source of confusion because the existence of alternative chromosomal and cytoplasmic states was central to its original usage. .... It now seems to us that the most meaningful biological distinction is between plasmids which promote conjugation, which we will classify as sex factors, and those which do not."

Chromosomal Associations The understanding of the possibility of the attachment (by some unknown mechanism, often diagramed as a "bump" on a linear diagram of a chromosome) of the F factor to the chromosome in Hfr strains probably helped the understanding of the linage of the fertility property and the genetic determinant for lactose fermentation (lac) in the work of F. Jacob and Edward A. Adelberg in 1959 (Compt. rend. acad. sci. 249:189). They concluded that the F-factor could become associated with cell genes which then became part of the "infectious hereditary particle" that was the F-factor. Soon these "augmented" F-factors became known as "F-prime" factors. Soon many variant F-primes were found and it was realized that F-prime plasmids carrying any desired part of the bacterial chromosome could be constructed. Elie Wollman recalled the history of F-prime factors (quoted in Thomas Brock, The Emergence of Bacterial Genetics, Cold Spring Harbor Press, 1990, p. 104): "Adelberg had brought back to Berkeley some of our Hfr strains. I spent the year 1958-59 in Berkeley -- finishing the writing of our book [Wollman and Jacob, 1959]. Once Ed Adelberg came to me telling me that one of the Hfr strains had changed: the frequency of recombinants was less than the expected, but all were donors of intermediate frequency. I suggested that, by comparison with HFT phage the sex factor had left its site accompanied by neighboring genetic fragments. This was verified experimentally. Lwoff, who had come to visit, brought the news back to Francois Jacob who immediately used it for making partial Lac diploids. This is the history of F prime factors."

The Physical Nature of Plasmids and Episomes: DNA By 1960 it was clear, of course, that "the genetic material is DNA", but the identification of cytoplasmic DNA was still questionable. Likewise, the structure of DNA in genes and in chromosomes was debated. The sizes of DNA "molecules" seemed to increase each year as the methods of preparation improved, and as the techniques for study of large, linear polymers got better. The recognition that shear forces could easily break large DNA molecules was especially important. Since bacteriophage were believed to be simple models for the genetic material of the cell, the nature of the DNA in phage was thought to be relevant. The sizes of the DNA in phages was rather ingeniously and indirectly determined by a technique known as "star gazing." This method compared the amount of DNA radioactivity (32-P) in a single phage particle, with the amount of radioactivity in the isolated DNA molecules released from the same phages under very gentle conditions. The radioactivity was detected by counting (under a microscope) the tracks in photographic emulsion in which the phages and the DNA were embedded. Each phage particle and each DNA molecule formed a "star" of such tracks. Since the number of tracks was the same for the intact phage particle and released DNA molecule, it was concluded that the DNA was present in the phage particle as one long piece (possibly held together by some non-DNA linkages). From the chemical composition of the phage and the bulk specific activity of the DNA, it was possible to calculate the molecular weight of the phage chromosome. That plasmids are DNA was rather conclusively demonstrated in physical experiments, first reported in 1961 by J. Mamur, R. Rownd, S. Falkow, L.S. Baron, C. Schildkraut and P. Doty (Proc. Natl. Acad. Sci. USA47: 972) who used the CsCl buoyant density separation of DNA based on nucleotide base composition to show that "light density E. coli-like DNA" appeared in Serratia marcescens (which has a somewhat "heavier" DNA) after transfer of the F-factor to Serratia. In 1962 S. Silver and H. Ozeki provided evidence for the same conclusion based on labeled DNA transfer of the colicine factor (Nature 195:875).

The "Campbell Model" Most experiments on the chemistry of DNA confirmed that DNA molecules were very long, linear, non-branched structures. How, then, to envision the attachment of episomes to the chromosome? In 1962, in a review on episomes (Adv. Genetics 11:101), Allan Campbell proposed a beautifully simple solution to this problem: the recombinational interaction of one circular molecule with another. [To view an excerpt from this reference Click Here]. "The Campbell Model" as it came to be known, explained the reversible association of some episomes with the chromosome, the inversion of the genetic map of lambda bacteriophage upon lysogeny as recently reported in 1960 by E. Calef and G. Licciardello (Virology 12:81), and the formation of double lysogens and defective heterogenotes in lambda phage (J. Whitfield and R. Appleyard, Virology 5:275, 1958). The apparently crucial insight of Campbell was that the episome must exist as a physical circular DNA structure. Interestingly he reasoned from the genetic map of phage T4 (there were, of course, no physical structures established for genomes). (p. 112) "Detailed linkage studies lead to the conclusion that the genome of one phage (T4) is indeed circular (Streisinger, Edgar and Harrar, quoted by Stahl (1961). If circularity is a property of phages in general, the equivalent of the insertion hypothesis is to the one circle out of two.[....] If the phage genome [now referring to lambda] is circular rather than linear, the lambda chromosome need not be split into parts [to account for map inversion in lysogens] but rather could be cut at a specific point on the circle when it lysogenizes. It is actually very simple (on paper) to insert a circular phage chromosome into a linear bacterial chromosome by reciprocal crossing over (Fig. 2)."

It is, of course, interesting to note that while Campbell based his argument on the T4 genome, which turns out to be linear although it has a circular map, and he applied it to lambda which turns out to have a linear map, but a circular intracellular form.

Circular DNA While the genetics of plasmids pointed the way to circular forms, the chemistry of DNA was just becoming clearer. The key step in the modern concept of the plasmid was the confirmation that DNA molecules can, and often do, exist as circular structures. The first confirmation of this fact came again, from the study of phage biology. In an attempt to study the smallest life form, biologists had been studying bacteriophages, and the smallest known phages were two related phages phi-X174 and S13. Robert Sinsheimer had shown that phi-X was unusual in that it contained the single-stranded form of DNA rather than the double helical DNA of the Watson-Crick model. Using the recently characterized nucleases with specificity for exonucleolytic attack coupled with hydrodynamic studies, W. Fiers and R. Sinsheimer asserted in 1962 (J. Mol. Biol.5:408- 434) that phi-X DNA was in the form of a small circular, double stranded DNA molecule. This precedent for circular DNA molecules was soon followed by the discovery in 1963 of:

(1) the cohesive ends of the DNA of bacteriophage lambda and its ability to form circles (called "folded molecules" at the time) by A. D. Hershey, E. Burgi, and L. Ingraham (Proc. Natl. Acad. Sci. USA 49:748). (2) the circular structure of the E. coli genome by autoradiography reported by J. Cairns (J. Mol. Biol. 6:208). (3) the evidence that the DNA from polyoma virus is circular by R. Dulbecco and M. Vogt (Proc. Natl. Acad. Sci. USA 50:236) and by R. Weil and J. Vinograd (Proc. Natl. Acad. Sci. USA 50:730)

Even though these studies with phage and viral DNAs provided the methods and concepts to characterize circular DNAs, the study of the physical nature of most plasmids was complicated by the difficulty in separation of the plasmid DNA from the mass of chromosomal DNA. This problem was solved in 1967 by the introduction of the dye-buoyant density method by R. Radloff, W. Bauer, and J. Vinograd (Proc. Natl. Acad. Sci. USA 57:1514). This method depended on the restriction on binding of a DNA-intercalating dye such as ethidium bromide by covalently closed circular DNA molecules in comparison to linear and nicked circular molecules. These dye-DNA complexes could be separated in density gradients of the dense salt CsCl formed in the ultracentrifuge. Plasmid DNAs were easily isolated by this method for detailed characterization and in 1968 M. Bazaral and D.R. Helinski applied this method to colicine factors E1, E2 and E3 and showed that these factors were circular DNA molecules of homogeneous molecular weights (J. Mol. Biol. 36:185). Beginning in 1959 (A.K. Kleinschmidt and R.K. Zahn, Zeitsch. Naturforsch. 14b:770-779, 1959) electron microscopic visualization began to be applied to DNA molecules spread in protein films, and this technique soon allowed "direct" visualization of both phage and plasmid DNAs and provided dramatic confirmation of the circular nature of plasmid DNAs.

R-factors Another important class of plasmids which were discovered in relation to their pathogenesis is the R-factor. In the early 1950s it was observed in Japan that multiple antibiotic resistance was developing in a single step in patients with enteric infections. In 1960 T. Akiba, T. Koyama, Y. Isshiki, S. Kimura, and T. Fukushima (Jap. med Wochschr. 1866:45) described these phenomena and in 1961 T. Watanabe and T. Fukasawa reported that this multiple drug resistance was being transferred by a plasmid (? an episome) which they called a resistance transfer factor (RTF or R-factor) (J. Bact. 81:679).

Organelle Genetics As difficult as it was to elaborate an understanding of the genetic and physical basis for non- chromosomal heredity in bacteria, the parallel history of eucaryotic cells is even more tortuous. While many observations in eucaryotes (mainly yeast and protozoans, single-cell organisms more amenable to genetic analysis than many multicellular organisms) suggested that non-chromosomal, especially cytoplasmic heredity exists, the acceptance of this conclusion and evidence for its physical basis were long in coming. Mitochondrial genetics, pioneered in the 1950s by B. Ephrussi and P. Slonimski in their studies of the respiratory-deficient petite mutants of yeast (such mutants grow slowly, depending as they do on glycolysis, and give small colonies, hence the designation, petite), became well-established only in the late 1960s when many additional mutants were identified that were associated with the mitochondria. Also, as early as 1954 some mutations in Chlamydomonas were found by R. Sager (Proc. Natl. Acad. Sci. USA 40:356-363) to behave in non-Mendelian fashion and were attributed to mutations in the chloroplasts. Finding the physical basis of organelle heredity (that is, the DNA in these structures) proved difficult as well. Cytochemical, electron microscopical, and biochemical evidence was offered, but until the techniques for study of DNA based on sequence comparisons (first nucleic acid hybridization and more recently direct nucleotide sequence analysis), the existence of cytoplasmic genes in eucaryotes was controversial. As R. Sager noted in 1972 (Cytoplasmic Genes and Organelles, Academic Press, p. 2):

"The pendulum of opinion had swung from one extreme -- cytoplasmic genes do not exist because we do not see cytoplasmic chromosomes to the other extreme -- cytoplasmic DNA's exist, and therefore there must be cytoplasmic genes."

The "Modern Period" of Plasmid Research By the end of the 1960s, then, both the genetic and physical understanding of plasmids and cytoplasmic heredity had reached a level of detail which was to allow the subsequent massive exploitation of these genetic elements as tools to study key cellular processes such as DNA replication as well as to manipulate and engineer the genetic contents of cells at will by means of the newly devised methods of in vitro recombinant DNA chemistry.

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