LOUIS PASTEUR (1822-1895)

The True Master of Microbiology

King-Thom Chung and Deam Hunter Ferris

King-Thom Chung is a professor of microbiology at the Department of Biology, The University of Memphis, Memphis, TN. Deam Hunter Ferris, Professor Emeritus of the Department of Pathobiology, University of Illinois, Urbana, IL., passed away on Dec. 8, 1993, during the preparation of this article. This article is dedicated to him.

It is fair to consider Louis Pasteur as one of the first, if not the first, "modern" microbiologist. His story is well known and we will not repeat it in detail, but we will focus upon the human side and the problems he overcame in helping establish microbiology as a science. Remember that he was a chemist---but a chemist with a difference!

Pasteur (the name means "shepherd") came from a humble beginning, his father being a tanner. After taking an extra year to go through the Normal School in Paris (Ecole Normale), he became interested in the lectures of the great chemist, Professor Jeane-Baptiste Dumas, and changed to a major in chemistry. Pasteur, unlike most chemists, had a need for the microscope as he was studying the crystals of tartaric acid. Upon graduation he continued work on crystals and at the age of 26 made a modest discovery--that there were 4 kinds of tartaric acid crystals instead of two. The microscope played an important part in this work, as the crystals looked alike, but were mirror images of each other. This discovery aroused the interest of leading chemists in France and he was made professor at Strasbourg and became a colleague of professors three times his age. He continued to work on crystals. He married and was promoted Professor and Dean of the Faculty of Sciences at Lille. This move was to become momentous for Pasteur and for the entire world. At Strasbourg, Pasteur was at work day and night on his beloved study of crystals. But then, at Lille, he was confronted by a committee of business men who told him bluntly that basic science may be all right, but they wanted to know if science could help business. They asked Pasteur if he would be able to raise the yield of sugar from sugar beets and increase the production of alcohol. Only a short time before this, Cagniard de la Tour had discovered

that the fermentation of beer took place by the action of yeasts. He published a clear paper, but it received little attention, and was not generally known, even by scientists.

A more direct challenge came when one distiller of alcohol came to Pasteur in great distress. His vats were filled with sugar beets seeded with yeast, but they would not produce alcohol. He was losing thousands of francs each day. Pasteur, the chemists with a microscope, took samples from the vats and studied them under the microscope. This would never have been done by an ordinary chemist and Pasteur's experience studying crystals under the microscope became a boon for humanity. What he saw was globules smaller than any of his tiny crystals. He remembered the papers of de la Tour and recognized that these were yeasts. Immediately he saw that de la Tour was right. The yeast was alive and produced the alcohol from beet sugar.

How to help the brewer? Pasteur found that in the "sick" vats there were no yeast cells, but he found something that was to start the field of microbiology! He found masses of tiny rods--and the vats with the rods had the smell of sour milk! He did some thinking-if the rods were alive, like the yeast cells in the healthy vats, perhaps they multiplied and caused the "sickness" of the beet-sugar fermentation. He realized further, that the rods needed what today we call a pure medium in which they could grow and he could study them in a pure culture. The development of a culture medium for the bacteria took time; he tried a sugar solution and other solutions with success, but finally developed a medium of the right pH and with adequate nutrients to sustain growth. All of this was accomplished in the midst of teaching and other heavy work. He attempted at this time to be elected to the Academy of Science and failed.

Pasteur worked day and night until he was able to grow pure cultures of the bacteria and the yeasts. But his work at Lille attracted no attention and he moved back to Paris where he became Administrator and Director of Scientific Studies at the Normal School. With great difficulty he again set up a tiny laboratory and went to work with his incubators and microscope. Here he became embroiled in a debate with the great chemists of the brewing industry in France and Germany. The leading chemists, such as Liebig did not believe de la Tour or Pasteur, who were saying that yeasts cause the fermentation which creates wine and beer. Liebig had insisted that it was albumin in the vats which produced the fermentation. Pasteur, after developing the methods of microbiology necessary for pure cultures, proper media, sterile equipment and transfer methods, then demonstrated in a succession of experiments that the wines and beers were the results of fermentation by yeasts. Pasteur called "Fermentation," "life without oxygen." He was not modest about his work.

Pasteur now gave papers and speeches which proved that it was not so much the efforts of men, but the work of millions of yeast cells which produced wine and beer. The great Liebig was defeated and Pasteur was finally given the credit he deserved. Pasteur continued to work with his bacteria and yeasts and developed increasingly better methods to handling them. One great problem was contamination from the air. This was quickly entangled with a great scientific problem, not yet resolved at that time. In spite of Spallanzani 's work, most scientists still believed in "Spontaneous Generation". The general belief was that all life from mice to the microbial forms spontaneously arose when there was moisture and warmth. A sealed cutlure which was boiled would show no life, but it was believed that air was needed for spontaneous generation of microbes. This was a serious problem for Pasteur in his numerous debates. An assistant, Balard, solved the problem when he suggested heating and drawing the air tube of a flask out into an "S" or similar curve so that incoming bacteria would be deposited on the curved walls of the tube. The media in these flasks did not become contaminated, but when inoculated with bacteria deposited on the tubing walls, a culture grew. This experiment convinced even the opposition and Pasteur won his debate on Spontaneous Generation: "All life from existing life."

Pasteur studied the wine industry and learned the types of microbes which spoiled wine in different ways, as well as the organisms which produced perfect wines of different types. He truly mystified experts in the industry by his ability to judge the type of spoilage in various wines (and also recognize good wine) with his microscope! Pasteur's work greatly benefitted the wine and beer industries and even the vinegar producers. The next problem he was asked to work on was a disease in silkworms, important in the south of France.

Pasteur had more difficulty when asked to help the silkworm industry where silkworms were killed by a disease called pebrine, because of black spots resembling pepper on the larva, or silkworms. His first analysis proved wrong. The second year, with the help of an assistant, Gernez, it was found that a parasite was the cause of the silkworm illness. He demonstrated the method of selecting silkworm moths free of the parasite and how to produce worms that gave splendid yields of silk.

Paul Lister, the famous English surgeon who devised antiseptic and then aseptic surgery techniques,wrote to Pasteur a letter thanking him for having demonstrated the principle of microbial disease. Pasteur was very pleased with this recognition from the medical profession. By now Pasteur had put all of Europe on the alert about microscopic life. He himself now hoped to invade the arena of medicine and study the relationship of his bacteria to human illness. But he was beaten by a German country doctor, Robert Koch. Koch turned the world upside down by his demonstration of the tubercle bacillis as the cause of tuberculosis. He then demonstrated that it was another germ which caused the dreaded disease of man and livestock ---anthrax. Ignatz Semmelweis, an Australian physician, had recently pronounced that child-bed fever was contagious and that the obstetric wards of Paris were "pest holes."6

At the meeeing of the Academy of Medicine in Paris, Pasteur broke up the meeting and scandalized the Academy by interrupting the prominent physician lecturing on the disease. Pasteur interrupted him loudly, and, told him bluntly that he was wrong about the cause of the disease, that the physicians themselves were carrying the infection. When the famous doctor told him that he would never find the microbes, Pasteur dragged his partially paralyzed leg to the rostrum and on the board drew a chain of small circles. "I have found it already and here is how it looks."

Pasteur was now in his fifties. He had been a chemist known for work on tartaric acid, then an expert on beet-sugar fermentation, the one who found the causes of wine spoilage and saved this industry. Pasteur coined the slogan "Better Beer for France" and had the goal of making France as famous as Germany for quality of its beer. He literally dashed from these tasks to save the silkwork industry. But all of this time he had the big goal of working with microbes that he knew caused human disease. He was somewhat shocked to realize that Koch had beat him to this. Pasteur considered that microbes were by now his rightful field and that he had demonstrated this when Koch was just a child.

There were human reasons why Pasteur had not gotten into the medical field. He knew nothing of anatomy and physiology and did not like the smell of hospitals. He was squeamish, also, about sticking guinea pigs with needles and really turned off, as we will see later, by the idea of biopsies and exposing the brain of an animal for direct inoculation. Fortunately, he took on three newly graduated medical doctors as assistants, Joubert, Roux and Chamberland.

With his assistants, Pasteur began to study anthrax, collecting and growing various strains. With these he checked out the efficacy of "cures" pronounced by veterinarians--all of which failed. But, some cows survived the disease, and Pasteur wisely saved and studied these animals. When injected with his most virulent strain of anthrax, the animals survived the challenge.

THE CONCEPT OF IMMUNITY

The concept of immunity was born! Between 1887-1890 Pasteur was working on chicken cholera and a dozen other animal maladies. At one time only very old cultures of chicken cholera germs were used to inoculate chickens. To Pasteur's surprize, they recovered. He then inoculated them with a fresh virulent strain. They survived challenge with a virulent strain--and he discovered a new principle!

THE CONCEPT of ATTENUATION

The "weakening" or attenuation of microbes for use as vaccines grew out of these experiments. Of course, the use of cow-pox as a vaccine for smallpox was known, but Pasteur realized that the use of a weakened microbe of the same species might be a superior method as in most diseases, a convenient weaker strain did not exist in another host.

PUBLIC EXPERIMENTAL DEMONSTRATION OF ANTHRAX VACCINE

There has probably never been a microbiologist and immunologist besides Pasteur willing to do a major experiment in public when failure could have cost his reputation, a reputation which had been made with such great difficulty and against so much prejudice. Pasteur was now in international confrontation with Koch, who discovered the anthrax bacillus as the cause of that killer of man and animal. Pasteur had just announced the protection of sheep with a strain of anthrax he had attenuated. His detractors were many and there was widespread doubt about the accuracy and validity of his work for veterinary medicine. He was not a veterinarian or physician. The well known Baron de la Rochette was persuaded by Pasteur's enemies to persuade him to make a large scale public experimental demonstration of his anthrax vaccine on 50 animals. This was a very costly experiment for the Society, but several prominent members wanted to bring Pasteur, the out-of-bounds chemists, down. They did not believe his lucky "magic" cures were real.

Pasteur was not a suspicious man, and, against the advice of his medical colleagues, assented at once. He silenced his associates by assuring them that "what worked on 14 sheep in our laboratory will work on fifty at the public experiment in Melun!" Pasteur and his associates went by train to Pouilly-le-Fort, where 48 sheeps, 2 goats and several cattle were on hand for the trial, open to the public. Pasteur marched past the crowd of scientists, Senators and spectators as well as a large number of newspaper reporters, including the most famous reporter of the London Times. Alcohol lamps were lighted and syringes flamed; animals were inoculated with anthrax which would kill mice but not guinea pigs--the attenuated strain. Twelve days later the trial was repeated with a strain which would kill guinea pigs, but not rabbits.

The animals survived the vaccinations and the time came for the final challenge with the virulent anthrax germs, at which time, unvaccinated animals were also injected. The crowd that came at the conclusion of the experiment made the previous ones seems like nothing! Physicians, veterinarians, high government officials and a huge crowd of newspaper reporters as well as a huge number of citizens were present. All of the vaccinated animals, each of which but a few days before had been given an enormous dose of deadly anthrax were well, frisky and eating. All but two of the unprotected animals were dead and the two were staggering with deadly ooze of blood from their noses and mouths.

Pasteur was now regarded as the miracle workers of the day. But this is unique in the history of microbiology and even science itself. No one before or since has carried out such a public experiment. Doctors of Medicine and Veterinary Medicine who had previously been most sarcastric and critical of Pasteur and his reports now came forward to congratulate him. The great reporters from the Times and other papers rushed to report that public experiment was a "perfect and unprecedented success."

Pasteur's little laboratory was now turned into a vaccine factory which today we would consider impossibly crude. Huge kettles were filled with the broth holding the tame anthrax and Pasteur's associates worked overtime to produce vaccines which would save the sheep industry. Modern microbiologists need to consider the concern of these microbiologists, ladling a few ounces of the bacillus-broth into glass bottles, simply clean and not really sterilized. They had to do this complex work without any adequate equipment. In less than a year hundreds of thousands of sheep had been protected by the efforts of this small laboratory.

PASTEUR AND "FILTRABLE VIRUS"

The genius of Pasteur and the growing skill of the new microbiology profession is no more manifest than in the story of rabies vaccination. Pasteur once said: "I have always been haunted by the cries of those victims of the mad wolf that came down the street of Arbois when I was a little boy." In France, the fear of rabies was so great that laws had to be passed to stop the shooting of those sad cases of fellow citizens suspected of dying from rabies.

In the midst of an international debate over the quality of his anthrax vaccine and critical papers from Koch--for there had been impure batches from those kettles, and some disastrous deaths of sheep--Pasteur plunged into a search for the causative agent of rabies. He personally took samples of saliva from the mouths of sick and vicious bull dogs whose sudden lunge and bite could have meant a horrible death. He also took samples from human victims and as often happened in his research, started out with a mistake. From one child he cultured an organism that he named "the microbes like an eight." He even read papers before the Academy of Science in which he postulated that his "figure-eight" germ might be the cause of the dreaded hydrophobia. Roux and Chamberlain, under Pasteur's guidance, soon found that the "figure-eight" bacterium was to be found in the mouths of normal children, also.

The dangerous job of capturing rabid dogs and allowing them to bite normal dogs was carried out in the laboratory. They also used the saliva of the mad dogs to inoculate laboratory animals. The results were strange. Some animals came down immediately while others waited weeks or months to succumb. Some research on rabies required that the virus be deposited directly on the brain of a dog. Pasteur was squeamish about opening up the skull of a dog and exposing the brain. Fortunately, Roux, one of his medical surgeon assistants--to whom this was merely a minor operation--did the procedure in Pasteur's absence from the lab. The experiment was a complete success and Pasteur allowed greater freedom to his qualified assistants.

However, unlike other diseases, Pasteur was not able to isolate a bacterium which clearly caused the disease. He was not able to find anything which would grow in or on any of his media. The genius of the microbiologist arose to the occasion. He asked, "was the rabies agent something which would pass a filter retaining all known bacteria?" It proved to be! A new concept arose: the FILTERABLE VIRUS---the secret here was to develop a filter with a known pore size, to find the cut-off point at which no "filterable" virus would pass the filter. In this way the microbiologists in Pasteur's laboratory learned that there were organisms too tiny for the resolving power of the light microscope, but nevertheless, particles, which could be filtered for purification and isolation. For years after this era, these microbes were called "filterable viruses."

From hundreds of dangerous experiments with dogs and other animals, the concept of how the rabies organism attacked the nervous system, finally reaching the brain, became established in Pasteur's mind. He and his assistants found a way to attenuate the virus by drying in a sterile bottle the spinal cord of an infected rabbit. By giving a series of 14 injections he was able to protect every rabid dog inoculated. None of the dogs injected with the attenuated rabies vaccine died. At one time he had the "modern" thought of vaccinating all dogs in France--but wisely decided he did not have the facilities to carry this program out. He undoubtly did not want to repeat the anthrax vaccine disasters.

From all over the world--even from Emperor of Brazil--there poured frantic letters asking for the vaccine for human beings who faced the sure death from rabies. But Pasteur held back from this ultimate test. Pasteur even considered seriously starting on himself; he wrote to a friend he thought that he would infect himself, then take the vaccine.

One day a woman from Alsace brought her a 9-year old boy into Pasteur's laboratory--Joseph Meister, who had been bitten in 14 places by a mad dog just the day before. She begged Pasteur to save her little boy. Pasteur brought in two physicians. When they saw the festering wounds they agreed that Pasteur should try his canine vaccine. If he did not the boy faced a certain death. On July, 1885, the first injection of the attenuated rabbit spinal cord vaccine into a human being was made. Little Joseph then went through the fourteen daily injections--really just pricks of the hypodermic needle under his skin. Joseph Meister went back to Alsace with no sign of disease. Pasteur's doubts vanished and he proclaimed the triumph to the world. The world immediately responded and a flood of humankind poured into the little Paris laboratory.

Russia even sent 19 peasants from Smolensk, all bitten by a mad wolf and some so mangled they could not walk. All the leader of the group knew in the French language was "Pasteur". It had been two weeks since the peasants had been bitten and Pasteur was fearful that few if any could be saved. But he changed his routine and gave them 2 injections per day. The vaccine saved all but three of the otherwise doomed Russians. Paris went wild and the Russians returned to a nations which greeted them with the same awe as one might have for a person raised from the dead. The Tsar of Russia sent Pasteur the diamond cross of St. Anne plus a hundred thousand francs to start building that became known as the Institute Pasteur.

One of the authors has visited the Pasteur Institute and also the house where Pasteur died as well as the small laboratory which contains writing on the wall by Pasteur himself. The author was permitted to use Pasteur's favorite microscope (The field of each lens was much smaller than those in use today, but the optical quality of each lens was superb.)

A BRIEF PERSONAL SKETCH

A little on the personal life of Pasteur might be of interest. Pasteur was a good man by all standards. He was born in a humble family on December 27, 1822. in a small town name Dole, just outside of east France. His father was named Jean-Joseph and mother Jeanne-Roqui. He had three sisters: Virginie, Josephine and Emilie. They grew up in Arbois, while Louis's father was a tanner, which is a family tradition for several generations.

As a child, Louis Pasteur loved the outdoors and would spend as much as time he could on long walks in the woods. He also liked fishing and was very fond of animals. He was also interested in working with his crayons by which he earned a reputation as an artist. This was his first interest. He was also interested in drawing. Although the Pasteur family was poor, Louis's parents paid a great deal of attention to his education. They had great hopes for Louis. His father was certain that his son would be more than a tanner. They made real sacrifices to provide for his schooling and were always genuinely interested in what he was learning and supportive in all his endeavours. Louis Pasteur was not quick in learning. He never affirmed anything unless he was sure of it.

At the age of 15 (Oct. 1838), Louis left home for school in Paris with his father's encouragement. This did not work out because Louis was terribly lonely and homesick. So he finished school at the College d'Arbois (near his home town), then got to the Royal College in Besancon, where he earned his Bachelor of Letters (1840 at the age of 18). There Louis learned that hard work will open the doors to success. In 1842, not quite 20 years old, Louis went to Paris again and took the entrance examination for the Ecole Normale, a university he had wanted to attend. Since he only ranked 15 out of 22 in an entrance examination. He did not want to enter with such a poor ranking. So, instead, he went at this time to the Pension Barbet, where Louis was strongly inspired by Professor Jean-Baptiste Dumas at the Sorbonne at this time. Louis became a disciple of Professor Dumas, who was the founder of organic chemistry and helped him get started with chemical research.

Louis Pasteur had two loves: one for his studies and the other for God. He was very close to his family and helped his sisters out every chances he got. His love was so great for his sisters that he offered his salary from Royal College (Besancon) to help them with their education. This was very forward thinking because at this period of time few women attended college. Louis was very thrifty himself. He hated to spent more than 50 cents on any meal and kept strict track of all his expenditures. He once had to buy a stove for his room because it was too cold to study, but he rented one instead, because it was cheaper.

Louis again attempted the test to enter Ecole Normale and scored 4th in his class. He now felt that he was ready to learn all he could learn. Classes at the Ecole was free and Louis took mathematics, chemistry and physics there. Louis also took classes at the Sorbonne. He took classes such as glass blowing, carpentry and metal working which were necessary as most chemists had to make their own equipment. He also took chemistry, mineralogy, and crystallography which became his favorite. Louis completed his studies for Bachelor of Science in 1847.

During the French Revolution in 1848, Louis was enthralled in the magic of it and had enlisted in the National Guard. This was against his parent's wishes. He never had to fight and soon the revolution was over. It was shortly after this that Louis made his discovery in crystals. Very soon after his discovery, Louis's mother died of apoplexy. He was greatly saddened because he did not make it home in time and he always wanted his parents to share in his accomplishments.

Louis met his wife Marie Laureut, the daughter of the president of the University of Strasbourg, where he had a teaching job in May of 1949. It was love at first sight for both of them, Louis was distracted. Louis had only known Marie for two weeks when he sent a letter to her father asking for her hand in marriage. The wedding took place on May 29, 1849 in the French cathedral St. Madeleine in Strasbourg. On the day of his wedding he was missing and they found him in the laboratory and brought him to the church. After his wedding he was found back in the laboratory and his wife was there by his side. Marie became his wife, friend, collaborator, secretary and mental supporter all his life. He was a good family man and enjoyed his family immensely. They had five children, four girls and one boy. Unfortunately, only two lived through their childhood.

At the age of 46, in 1868, Louis suffered a paralytic stroke and almost died. He recovered but walked with a limp and did not have the use of his right hand. In 1887, Pasteur had another stroke that prevented him from being able to do anything experimental. Perhaps these events were too much for his work-weakened body. Years before, Pasteur had had a small house built near the kennels where the rabid dogs were kept. One author has visited this place and seen where Pasteur marked the heights of his wife and children on a door. He often took a "vacation" in this house where he could be in a small laboratory near the kennels. Here he died on 5 o'clock in the morning of September 28, 1895. Before his death, he said " peace, love and science!". He was surrounded by his family. France wanted to bury Pasteur in a Pantheon reserved for its greatest heros, but his family insisted on his burial at the Pasteur Institute where he rest in a beautifully designed tomb.

Pasteur was a devout Catholic, and died with a crucifix in his hand. Pasteur was a believer. All his life he followed what he considered was his God-given mission to save life from disease. We all are benefited by Pasteur's scientific contribution Without Pasteur, we are not sure all of us would be living today. His life serves as an good example of a truly good man. His love of his family, friends, country, sincere honesty to truth and devoted hard working spirit exemplify the highest human virtues He seemed to possess the spirit of universal truth. He is truly a "perfect man of all ages" which all microbiologists seek to emulate.