Loeffler (Löffler), Friedrich August Johannes

(b. Frankfurt an der Oder, Germany, 24 June 1852;d. Berlin, Germany, 9 April 1915)

microbiology, medicine,

Loeffler’s father, Gottfried Friedrich Franz Loeffler, was a distinguished physician who rose to the rank of Generalarzt in the Prussian army. He wrote books on military medicine and from 1867 until his death in 1874 was assistant director of the Friedrich Wilhelm Institut for military doctors in Berlin, where he did much to raise the status of army medical officers. With this advantage the younger Loeffler began his own studies at the French Gymnasium of Berlin and became fluent in French, an important skill for those who were then pursuing microbiology. He attended the medical school of the University of Würzburg before transferring to his father’s school just before the Franco-Prussian War. After serving as a hospital assistant in that conflict, he was awarded the medical degree in 1874. Loeffler then became assistant physician at the Charité Hospital in Berlin, where for a year and a half he came in contact with some of the best clinicians of the time. From 1876 to 1879 he served as military surgeon and public health officer in Hannover and Potsdam, and in October 1879 he was assigned to the newly established Kaiserliches Gesundheitsamt in Berlin.

The assignment proved to be the turning point of his career. Nine months later Robert Koch arrived to set up a bacteriological laboratory, which, modest at first, soon became a leading center of research and discovery. Already famous for his work on anthrax, the development of a solid medium for cultivating bacteria, and a monograph on wound infection, Koch chose Loeffler and Georg Gaffky as his assistants. The next four years were to be highly productive for all the members of Koch’s group. As Loeffler later recalled:

The memory of those days, when we still worked in this room, Koch in the center and we about him, when almost daily new wonders in bacteriology arose before our astounded vision, and we, following the brilliant example of our chief, worked from morning to evening and scarcely had regard to our bodily needs—the memory of that time will remain unforgettable to us. Then it was that we learnt what it means to observe and work accurately and with energy to pursue the problem laid before us [in F. Nuttall, “Biographical Notes” 235].

During Loeffler’s four years at the imperial health office laboratories he pursued a number of bacteriological problems, including a series of experiments to determine effective means of disinfection. A significant practical result of these studies was the identification of the infectious agent of glanders, a disease seen mainly in horses. With the identification of the bacterium came the practical consequence of prevention. Although several others had described the occurrence of microorganisms in cases of glanders, no positive identification of an etiological agent had been made.

As with most of Loeffler’s subsequent bacteriological work, the glanders studies were models of the new experimental methods and criteria as applied to bacteriology by Koch. Loeffler took materials from glanders nodules in the lung and spleen of a horse and cultured them in test tubes containing blood serum. Here the bacteria were successfully grown in succeeding generations and then inoculated into a healthy horse, which began to show typical symptoms of glanders within forty-eight hours. At autopsy Loeffler found fresh nodules which again were productive of bacteria. He now clearly demonstrated them by staining with methylene blue. These bacteria, injected into rabbits, mice, and guinea pigs, produced the signs and symptoms suggestive of glanders. Bacilli cultivated from the small experimental animals, when injected into horses, produced the typical disease of glanders. Thus, in this sequence of experiments Loeffler satisfied the so-called Koch’s postulates, in 1882, the year Koch enunciated them: to isolate the organism, to grow it in pure culture, to infect experimental animals with the cultured organism, and then to again isolate the bacteria from the experimental animals.

Loeffler’s best-known work is the elucidation of the characteristics of the diphtheria bacillus and its growth in pure culture (1884), which again clearly revealed the imprint of Koch, with whom he was then so closely associated. For the first time bacteriologists could work with single microbial species even though the original specimen taken from the throat of a patient, for instance, might be teeming with myriads of organisms of different species.

Diphtheria, a disease known since antiquity, to the history of which Loeffler also devoted much time, was particularly feared because it produced a false membrane in the throat that could suffocate its victims, especially children. In 1871 Max Oertel, of Munich, showed that the false membrane could be produced in rabbits by swabbing their throats with secretions from human patients. In 1875 Edwin Klebs postulated a fungus as the cause, but at the German Medical Congress of 1883 Klebs presented new information pointing to a specific bacterium that could be seen, after staining, in the throat membranes of diphtheria patients. The task remained to differentiate the several bacteria that were implicated in the disease and to grow in pure culture the one responsible for causing it.

One of the difficulties Loeffler faced in isolating the agent of diphtheria was that the throats of diphtheria patients carried many microorganisms, one of which, the Streptococcus, had already led to much confusion. In a series of twenty-seven cases of fatal throat inflammation, twenty-two had been diagnosed as diphtheria, five as scarlatinal diphtheria. In the latter, Loeffler found that the Streptococcus was the dominant organism. It is now known that scarlet fever is accompanied or preceded by a streptococcal throat infection. In the case of diphtheria, Loeffler reasoned that these chains of cocci played a secondary role.

In the case of typical diphtheria Loeffler observed that the bacteria described by Klebs were easily demonstrated in about half the cases he studied. Loeffler found these bacilli, which stained markedly with methylene blue, in the deeper layers of the false membrane but never in the deeper tissues or other internal organs, although these organs may have been greatly damaged. Loeffler still had to culture both the Klebs bacillus, never grown before, as well as the Streptococcus to prove or disprove either one as the cause of diphtheria. The Streptococci were easily grown on the solid medium of peptone and gelatin devised by Koch. Inoculation into animals produced generalized infections but never a disease resembling human diphtheria.

The bacillus implicated by Klebs—and now strongly suspected by Loeffler as well—as the diphtheria-causing organism was difficult to culture on the usual gelatin plates because it would not grow at the low temperatures required to keep the gelatin solid. The Streptococci, on the other hand, grew well at temperatures below 24°C, needed to keep the medium from liquefying. At this point Loeffler’s innovative and experimental skills come out most clearly. He developed a new solid medium using heated blood serum rather than gelatin as the means of solidifying. This medium could now be incubated at 37°C, or body temperature. The Klebs bacilli grew well under these conditions. When they were injected into animals, Loeffler found that the guinea pig developed tissue lesions very similar to those of human diphtheria. Bacilli could be easily recovered from the infection produced at the site of inoculation, but they were never recovered from the damaged internal organs. Loeffler thus postulated that this, too, was similar to human diphtheria, in which the bacteria were confined to the throat membrane. He reasoned that perhaps the bacteria released a poisonous substance that reached other parts of the body through the bloodstream. This supposition was soon proved correct by the work of Émile Roux and Yersin, who did much to reveal the nature of the diphtheria toxin. In practical terms the toxin theory soon bore fruit in the work of Behring and others who developed an effective antitoxin to counter the effects of the soluble poison produced by the bacillus.

One further test carried out by Loeffler in this series of experiments to identify and isolate the agent of diphtheria was an attempt to culture the organisms from healthy children. Much to his surprise he was able to isolate the bacillus from one of the twenty subjects under study. He thereby called attention to the fact that not all people infected by the diphtheria bacillus or the tubercle bacillus had the disease diphtheria or tuberculosis. This concept of a healthy carrier had immense public health significance, especially in the period when medicine was making a headlong rush to ascribe all diseases to bacterial agents and when physicians too often simply equated the presence of a bacillus with a particular disease. The host factors thus had to come under study as well.

With the publication in 1884 of these wide-ranging studies of diphtheria, which in their process revealed not only the identifiable agent but the equally important phenomena of toxin production and the healthy-carrier state, Loeffler may be considered the principal discoverer of the diphtheria bacillus. He thus capped the findings of numerous clinical investigators of diphtheria and those of Klebs, who first observed the bacteria.

In 1884 Loeffler was transferred from the laboratory at the Gesundheitsamt to become director of the hygienic laboratory at the First Garrison Hospital in Berlin, where he continued his bacteriological studies and lectured on sanitation. At this time he discovered the cause of swine erysipelas and of swine plague. In 1886 he was appointed to the Faculty of Hygiene at the University of Berlin, and the following year, with his bacteriological colleagues, R. Leuckart and O. Uhlworm, he founded the Centralblatt für Bakteriologie und Parasitenkunde (later the Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheitenund Hygiene), one of the most influential journals in the field.

In 1888 Loeffler, renowned as a bacteriologist, researcher, and teacher, received calls to chairs in hygiene at the universities of Giessen and Greifswald. He accepted the latter offer and spent twenty-five years as a distinguished professor, serving as rector of the university from 1903 to 1907. In 1905 he was raised to the rank of Generalarzt in the army.

Early in his years at Greifswald, Loeffler began work on the problem of mouse typhoid and its bacteriological cause. Salmonella typhi-murium. This led to the first attempt to use bacteria for controlling the spread of an unwanted animal population, Pasteur had earlier suggested this be done to reduce the rabbit population in Australia, Having learned of Loeffler’s paper describing a new bacteriological method for controlling field mice, in 1892 the Greek government asked him to assist it with control of the rodents that threatened the harvests on the plain of Thessaly. In his laboratory studies Loeffler had shown that Salmonella typhi-murium was infectious for mice but not for other farm animals, and he planned to spread the disease among mice by contaminating their food sources. With Rudolf Abel, his assistant at the university, Loeffler journeyed to Greece in April 1892, After determining that the Thessalian vole was as susceptible to the bacteria as his laboratory animals, he began to culture huge quantities of the Salmonella necessary to spread in the fields. The dispersed bacteria killed many field mice, but there was some debate as to the real, practical effectiveness of Loeffler’s efforts at biological control. The subsequent realization that the bacteria may not be innocuous to man precluded further study.

In 1897 Loeffler became chairman of a German commission to investigate foot-and-mouth disease of cattle. The following year, working with his assistant, Paul Frosch, Loeffler made yet another fundamental discovery. The first real proof of the existence of a pathogenic organism so small that it was invisible under the microscope came from the work of Dmitry Ivanowsky, who in 1892 demonstrated its activity in tobacco mosaic. The first knowledge that these agents, known as filterable viruses because they passed through porcelain filters that stopped the larger bacteria, were also capable of causing disease in animals resulted from the work of Loeffler and Frosch (1898). They discovered that aphthous fever (foot-and-mouth disease) in cattle was caused by such a virus.

Lymph from infected animals, diluted and filtered so as to be entirely free of bacteria, produced the typical disease after injection into test animals. The question now was whether the lymph contained an effective toxin or a previously undiscovered agent. Loeffler and Frosch believed the latter possibility, that the activity of the lymph in causing further disease was due to an organism that could multiply yet was invisible with existing microscopes. Shortly after their work became known, William H. Welch in Baltimore called it to the attention of Walter Reed, who soon elucidated the viral nature of yellow fever and its transmission by mosquitoes.

Through his discoveries of several microorganisms as well as through his teaching and methods of work, Loeffler contributed greatly to the so-called golden age of bacteriology. Besides his improved culture media, bacteriological technique is also indebted to him for the popularization of the alkaline methylene blue stain as well as a specific stain for cilia and flagella. He introduced the standard medium for cultivation of the typhoid bacillus and improved the methods of isolating and differentiating the numerous fecal bacteria. He is also known for work on disinfect ion, hygiene of milk and water, and sewage disposal.

In 1913 Loeffler was recalled to Berlin to succeed Gaffky as director of the lnstitut für Infektionskrankheiten. He was active at the beginning of World War I in planning hygiene programs for the army. His health began to fail in December 1914, and despite surgical intervention he died on 9 April 1915. He was buried in Greifswald.

BIBLIOGRAPHY

I. Original Works. Loeffler’s memoir on the diphtheria bacillus is “Untersuchungen über die Bedeutung der Mikroorganismen für die Entstehung der Diphtherie,” in Mittheilungen am dem kaiserlichen Gesundheitsamt, 2 (1884), 421-499; see also “Berichte der Kommission zur Erforschung des Maul und Kanlenseuche,” in Centralblatt für Bakteriologie und Parasitenkunde,23 , Abt. I (1898), 371-391, written with Paul Frosch. His many other medical and bacteriological papers appeared mainly in the Mittheilungen am dem K. Gesundheitsamte, the Deutsche medizinische Wochenshrift, and the Centralblatt für Bakteriologie. One of the first books dealing with the history of bacteriology was his Vorlesungen über die geschichtliche Entwicklung der Lehre von den Bacterien (Leipzig, 1887).

Loeffler’s papers available in English trans, are “On the Bacillus of Glanders”; “Investigations as to the Significance of Microorganisms in the Production of Diphtheria in Man”; and “Disinfection by Steam,” written with R. Koch and G. Gaffky, in W. Watson Cheyne, ed., Recent Essays by Various Authors on Bacteria in Relation to Disease (London, 1886); and “The History of Diphtheria,” in G. H. F. Nuttall and G. S. Graham-Smith, eds., The Bacteriology of Diphtheria(Cambridge, 1908), pp. 1-52.

II. Secondary Literature. The following works (listed chronologically) contain useful biographical data; F. Goldschmidt, “Die Begründer der modernen Diphtheriebehandlung,” in Münchener medizinische Wochenschrift, 42 (1895), 335-336; and “Professeur Friedrich Loeffler” in Revue d’hygiène, 22 (1900), 652-655; G. Gaffky, “Friedrich Loeffler,” in Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, 76 (1915), 241-245; George H. F. Nuttall, “Biographical Notes Bearing on Koch, Ehrlich, Behring, and Loeffler …,” in Parasitology, 16 (1924), 214-238; Dexter H. Howard, “Friedrich Loeffler and the Thessalian Field Mouse Plague of 1892,” in Journal of the History of Medicine and Allied Sciences,18 (1963), 272-281; and H. A. Lechevalier and M. Soltorovsky, Three Centuries of Microbiology (New York, 1965), pp. 122-136, 282-286.

Gert H. Brieger

Diphtheria

Diphtheria is a potentially fatal, contagious bacterial disease that usually involves the nose, throat, and air passages, but may also infect the skin. Its most striking feature is the formation of a grayish membrane covering the tonsils and upper part of the throat.

Like many other upper respiratory diseases, diphtheria is most likely to break out during the winter months. At one time it was a major childhood killer, but it is now rare in developed countries because of widespread immunization . Since 1988, all confirmed cases in the United States have involved visitors or immigrants. In countries that do not have routine immunization against this infection, the mortality rate varies from 1.5% to 25%.

Persons who have not been immunized may get diphtheria at any age. The disease is spread most often by droplets from the coughing or sneezing of an infected person or carrier. The incubation period is two to seven days, with an average of three days. It is vital to seek medical help at once when diphtheria is suspected, because treatment requires emergency measures for adults as well as children.

The symptoms of diphtheria are caused by toxins produced by the diphtheria bacillus, Corynebacterium diphtheriae (from the Greek for "rubber membrane"). In fact, toxin production is related to infections of the bacillus itself with a particular bacteria virus called a phage (from bacteriophage ; a virus that infects bacteria). The intoxication destroys healthy tissue in the upper area of the throat around the tonsils, or in open wounds in the skin. Fluid from the dying cells then coagulates to form the telltale gray or grayish green membrane. Inside the membrane, the bacteria produce an exotoxin, which is a poisonous secretion that causes the life-threatening symptoms of diphtheria. The exotoxin is carried throughout the body in the bloodstream, destroying healthy tissue in other parts of the body.

The most serious complications caused by the exotoxin are inflammations of the heart muscle (myocarditis) and damage to the nervous system. The risk of serious complications is increased as the time between onset of symptoms and the administration of antitoxin increases, and as the size of the membrane formed increases. The myocarditis may cause disturbances in the heart rhythm and may culminate in heart failure. The symptoms of nervous system involvement can include seeing double (diplopia), painful or difficult swallowing, and slurred speech or loss of voice, which are all indications of the exotoxin's effect on nerve functions. The exotoxin may also cause severe swelling in the neck ("bull neck").

The signs and symptoms of diphtheria vary according to the location of the infection. Nasal diphtheria produces few symptoms other than a watery or bloody discharge. On examination, there may be a small visible membrane in the nasal passages. Nasal infection rarely causes complications by itself, but it is a public health problem because it spreads the disease more rapidly than other forms of diphtheria.

Pharyngeal diphtheria gets its name from the pharynx, which is the part of the upper throat that connects the mouth and nasal passages with the larynx. This is the most common form of diphtheria, causing the characteristic throat membrane. The membrane often bleeds if it is scraped or cut. It is important not to try to remove the membrane because the trauma may increase the body's absorption of the exotoxin. Other signs and symptoms of pharyngeal diphtheria include mild sore throat, fever of 101–102°F (38.3–38.9°C), a rapid pulse, and general body weakness.

Laryngeal diphtheria, which involves the voice box or larynx, is the form most likely to produce serious complications. The fever is usually higher in this form of diphtheria (103–104°F or 39.4–40°C) and the patient is very weak. Patients may have a severe cough, have difficulty breathing, or lose their voice completely. The development of a "bull neck" indicates a high level of exotoxin in the bloodstream. Obstruction of the airway may result in respiratory compromise and death.

The skin form of diphtheria, which is sometimes called cutaneous diphtheria, accounts for about 33% of diphtheria cases. It is found chiefly among people with poor hygiene . Any break in the skin can become infected with diphtheria. The infected tissue develops an ulcerated area and a diphtheria membrane may form over the wound but is not always present. The wound or ulcer is slow to heal and may be numb or insensitive when touched.

The diagnosis of diphtheria can be confirmed by the results of a culture obtained from the infected area. Material from the swab is put on a microscope slide and stained using a procedure called Gram's stain . The diphtheria bacillus is called Gram-positive because it holds the dye after the slide is rinsed with alcohol. Under the microscope, diphtheria bacilli look like beaded rod-shaped cells, grouped in patterns that resemble Chinese characters. Another laboratory test involves growing the diphtheria bacillus on Loeffler's medium.

The most important treatment is prompt administration of diphtheria antitoxin. The antitoxin is made from horse serum and works by neutralizing any circulating exotoxin. The physician must first test the patient for sensitivity to animal serum. Patients who are sensitive (about 10%) must be desensitized with diluted antitoxin, since the antitoxin is the only specific substance that will counteract diphtheria exotoxin. No human antitoxin is available for the treatment of diphtheria.

Antibiotics are given to wipe out the bacteria, to prevent the spread of the disease, and to protect the patient from developing pneumonia . They are not a substitute for treatment with antitoxin. Both adults and children may be given penicillin , ampicillin, or erythromycin. Erythromycin appears to be more effective than penicillin in treating people who are carriers because of better penetration into the infected area. Cutaneous diphtheria is usually treated by cleansing the wound thoroughly with soap and water, and giving the patient antibiotics for 10 days.

Universal immunization is the most effective means of preventing diphtheria. The standard course of immunization for healthy children is three doses of DPT (diphtheria-tetanuspertussis) preparation given between two months and six months of age, with booster doses given at 18 months and at entry into school. Adults should be immunized at 10-year intervals with Td (tetanus-diphtheria) toxoid. A toxoid is a bacterial toxin that is treated to make it harmless but still can induce immunity to the disease.

Diphtheria patients must be isolated for one to seven days or until two successive cultures show that they are no longer contagious. Because diphtheria is highly contagious and has a short incubation period, family members and other contacts of diphtheria patients must be watched for symptoms and tested to see if they are carriers. They are usually given antibiotics for seven days and a booster shot of diphtheria/tetanus toxoid.

Reporting is necessary to track potential epidemics , to help doctors identify the specific strain of diphtheria, and to see if resistance to penicillin or erythromycin has developed. In 1990, an outbreak of diphtheria began in Russia and spread within four years to all of the newly independent states of the former Soviet Union. By the time that the epidemic was contained, over 150,000 cases and 5000 deaths were reported. A vast public health immunization campaign largely confined the epidemic by 1999.

See also Bacteria and bacterial infection; Epidemics, bacterial; Public health, current issues

Loeffler, Friedrich August Johannes (1852-1915)

German physician

Friedrich August Loeffler was a German physician who turned his career path to focus on microbiology after becoming an assistant to Robert Koch . Loeffler is accredited with the discovery of several microorganisms including Loefflerella mallei, the etiological agent of glanders, Corynebacterium diphtheriae, the infectious organism of diphtheria ; and Erysipelothrix rhusiopathiae, the infectious agent that causes cholera in swine. In addition to his discoveries of bacteria , Loeffler determined that foot-and-mouth disease was due to an infectious microorganism smaller than any bacteria (a virus).

Friedrich Loeffler began his studies in medicine at the University of Würzburg but then transferred to the Army Medical School shortly before the Franco-Prussian War. In 1872, Loeffler received his medical degree and then worked as an assistant physician in Berlin at the Charté Hospital. Beginning in 1876, he worked as a public health officer and military surgeon in Potsdam and Hannover. This lasted until 1879, when he moved to Berlin and continued his work at the Kaiserliches Gesundheitsamt.

Friedrich Loeffler's transfer brought him under the supervision of Robert Koch. Loeffler and Georg Graffky began assisting Koch on his research of bacteria. Loeffler first began his bacteriological studies researching effective methods of disinfection . During his studies, Loeffler discovered Loefflerella mallei, bacteria that causes glanders, a disease seen mainly in horses. To determine the exact bacteria that causes glanders, Loeffler applied what has come to be known as Koch's postulates . Initially, Loeffler isolated the infectious agent from the horse and grew it in a pure culture of blood serum. Next, Loeffler injected the bacteria into healthy horses, which then showed symptoms of the disease. Finally, Loeffler once again isolated the bacteria from the once healthy horses. In addition to discovering Loefflerella mallei, Loeffler discovered the infectious agent that causes cholera in swine.

In 1884, after a long struggle to decipher the etiological agent that causes diphtheria, Loeffler isolated Corynebacterium diphtheriae in pure culture from the throat of humans. Problems pinpointing the exact microorganism that causes diphtheria stemmed from the fact that many different microorganisms inhabited the throats of diphtheria patients. Loeffler undertook the task of isolating pure cultures of bacteria to determine the exact etiological agent of diphtheria. Loeffler found that certain throat infections were due to streptococcal infections, which are now known to cause scarlet fever. He reasoned that the Streptococcus bacteria were not responsible for causing diphtheria because when injected into healthy animals, the bacteria did not produce symptoms characteristic of the disease. Additionally, the Streptococcus was not always abundant in diphtheria patients. The Streptococcus appeared to be secondary to rod shaped bacteria. When these rod shaped bacteria, called bacillus, were isolated in pure culture and injected into healthy animals, the animals exhibited the characteristic signs of human diphtheria, including the pseudomembrane in the throat of the patients that suffocate to death. Additionally, Loeffler determined that toxins produced by the infectious agent were the cause of destruction to internal organs. He reasoned that the toxins released entered the blood stream and traveled to other organs thereby poisoning them. Emile Roux and Yersin proved this theory of toxins to be correct. Loeffler made a surprising discovery when he was able to isolate the diphtheria bacillus from healthy individuals. He then determined that not all people who carry infectious microorganisms contract the disease.

Also in 1884, Loeffler began his new career as hygienic director with his first directorship position being at the First Garrison Hospital. There he was able to continue his bacteriological research in conjunction with lecturing on sanitation. Two years later, he became part of the faculty at the University of Berlin. By 1888, he had transferred to the University of Greifswald where he spent the next 25 years.

At the University of Greifswald, Loeffler studied Salmonella typhi-murium, the bacteriological agent that causes mouse typhoid but does not infect other animals. This research was intended to control the exuberant mouse population that was threatening to destroy the crops of Greece. Loeffler effectively killed the mice by contaminating their food sources with the bacteria.

In 1898, Friedrich Loeffler, in conjunction with Paul Frosch, determined a filterable agent proving smaller than any bacteria previously discovered caused foot-and-mouth disease. This was the first hint that viruses existed. At that time, Loeffler was working at the University of Greifswald as head of the department of hygiene . Loeffler moved his laboratory to the island of the Insel Riems in order to safely continue his research on the disease. In 1913, Loeffler's research took a back seat to his new position as director of the Robert Koch Institute in Berlin. Once World War I began, all research on the Insel Riems ceased. Loeffler worked for the army to implement proper hygiene regimens until his death in 1915.

See also Coryneform bacteria; Streptococci and streptococcal infections

Roux, Pierre-Paul-Émile (1853-1933)

French physician and bacteriologist

Soon after becoming a doctor, Émile Roux began doing research on bacterial diseases for Louis Pasteur . It has taken a century, however, for Roux's contribution to Pasteur's work—specifically his experiments utilizing dead bacteria to vaccinate against rabies—to be acknowledged. Roux is also credited, along with Alexandre Yersin, with the discovery of the diphtheria toxin secreted by Corynebacterium diphtheriae and immunization against the disease in humans. Both colleague and close friend to Pasteur, Roux eventually became the director of the Pasteur Institute in Paris.

Roux began his study of medicine at the Clermont-Ferrand Medical School in 1872. In 1874 Roux moved to Paris where he continued his studies at a private clinic. In 1878 he helped create lectures on fermentation for Emile Duclaux at the Sorbonne, Paris. Duclaux introduced Roux to Louis Pasteur, who was then in need of a doctor to assist with his research on bacterial diseases.

In 1879 Roux first began assisting Pasteur on his experiments with chicken cholera. The cholera bacillus was grown in pure culture and then injected into chickens, which would invariably die within 48 hours. However, one batch of culture was left on the shelf too long and when injected into chickens, failed to kill them. Later, these same chickens—in addition to a new group of chickens—were injected with new cultures of the cholera bacillus. The new group of chickens died while the first group of chickens remained healthy. Thus began the studies of the attenuation of chicken cholera.

In the 1880's Pasteur and Roux began research on rabid animals in hopes of finding a vaccine for rabies . Pasteur proceeded by inoculating dogs with an attenuated (weakened) strain of the bacteria from the brain tissue of rabid animals. Roux worked on a similar experiment utilizing dead rather than weakened bacteria from the dried spinal cords of infected rabbits.

On July 4, 1885, a 9-year-old boy named Joseph Meister was attacked on his way to school and repeatedly bitten by a rabid dog. A witness to the incident rescued Meister by beating the dog away with an iron bar; the dog's owner, Theodore Vone, then shot the animal. Meister's wounds were cauterized with carbolic acid and he was taken to a local doctor. This physician realized that Meister's chance of survival was minimal and suggested to Meister's mother that she take her son to Paris to see Louis Pasteur, who had successfully vaccinated dogs against rabies. The vaccine had never been tried on humans, and Pasteur was reluctant to give it to the boy; but when two physicians stated that Meister would die without it, Pasteur relented and administered the vaccine.

Pasteur stated that he utilized the attenuated strain of the vaccine; his lab notes, however, confirm that he treated Meister with the dead strain that Roux had been working on. (Why Pasteur maintained that he used his attenuated strain is not clear.) In any case, Meister received 13 shots of the rabies vaccine in the stomach in 10 days and was kept under close observation for an additional 10 days. The boy survived and became the first person to be immunized against rabies.

In 1883 Roux became the assistant director of Pasteur's laboratory. He undertook administrative responsibilities to help establish the Pasteur Institute, which opened in 1888 with Roux serving as director (from 1904) and teaching a class in microbiology.

Also in 1883 Roux and Yersin discovered the diphtheria toxin secreted by Corynebacterium diphtheriae. The two scientists filtered the toxin from cultures of the diphtheria bacterium and injected it into healthy laboratory animals. The animals exhibited the same symptoms (and eventual death) as those infected with the bacterium. Other data to support their discovery of the diphtheria toxin included urine obtained from children infected with the microorganism. Toxin excreted in the urine was sufficient to produce the same symptoms of the disease in laboratory animals. In 1894 Roux and Louis Martin began to study the immunization of horses against diphtheria in order to create a serum to be used in humans. The outcome of their research led them to successfully treat 300 children with the serum.

Beginning in 1896 Roux researched different aspects of diseases such as tetanus , tuberculosis , bovine pneumonia , and syphilis until he became the director of the Pasteur Institute in 1904. At that time Roux ceased all personal research and focused solely on running the Pasteur Institute until his death from tuberculosis in 1933.

See also Bacteria and bacterial infection; History of microbiology; History of public health