History of Microbiology

HISTORY OF MICROBIOLOGY

VOCABULARY

Fermentation: the enzymatic degeneration of carbohydrates in which the final electron acceptor is an organic molecule (contains carbon). For example, ATP is synthesized by phosporylation (adding phosphate) and oxygen it is not required. Fermentation is the process that yeasts use to convert sugars to alcohol in the absence of air. Pasteurization: the process of mild heating to kill particular spoilage microorganisms or pathogens.  Aerobic: Requires environment with oxygen  Anaerobic: Requires environment without oxygen  Facultative: Can live in environments with and without oxygen.

MICROBIOLGY HISTORIANS

Aristotle (384 B.C.) Before Aristotle, people believed generally believed that diseases were caused only by God. He was one of the greatest scientists of his time. He theorized that ‘a vital force’ forms life. He noticed that mice were commonly found in barns where grain was stored. He thought that the mice grew from the grain and hay, and he coined the term “Spontaneous generation”, the hypothesis that living organisms arise from nonliving matter. As a matter of fact, he published a recipe that anyone could use to grow their own mice: darkness + hay + grain = mice. No one doubted this for more than a thousand years. Eventually, a new theory arose called the Germ Theory (disease is caused by germs), later known refined to the term “biogenesis”. Biogenesis is the hypothesis that living organisms arise from pre-existing life. For many years it was debated as to which of these hypotheses was correct.

Virgil (40 A. D.) He agreed with Aristotle about spontaneous generation. He theorized that bees grew from honey and that flies grew from meat. Again, this was accepted for centuries.

The Romans (100 A.D.) During the 1st century AD (year 100), glass had been invented and the Romans were looking through the glass and testing it. They experimented with different shapes of clear glass and one of their samples was thick in the middle and thin on the edges. They discovered that if you held one of these “lenses” over an object, the object would look larger.

These lenses were not used much until the end of the 13th century when spectacle makers were producing lenses to be worn as glasses.

The early simple “microscopes” which were really only magnifying glasses had one power, usually about 6X - 10X. One thing that was very common and interesting to look at was fleas and other tiny insects. These early magnifiers were hence called “flea glasses”.

1 Zacharias Janssen (590) He was a Dutch spectacle maker, and started experimenting with these lenses. He put several lenses in a tube and made a very important discovery. The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself! He had just invented the compound microscope (which is a microscope that uses two or more lenses).

Galileo (1610) He heard of their experiments and started experimenting on his own. He described the principles of lenses and light rays and improved both the microscope and telescope. He added a focusing device to his microscope and of course went on to explore the heavens with his telescopes.

Antoni Van Leeuwenhoek (Dutch parisitologist; 1673 - 1723) He is called the Father of Microscopy, but he did not invent the first microscope. He became very interested in lenses while working with magnifying glasses in a dry goods store. He used the magnifying glass to count threads in woven cloth. He became so interested that he learned how to make lenses. By grinding and polishing, he was able to make small lenses with great curvatures. These rounder lenses produced greater magnification, and his microscopes were able to magnify up to 270x. He decided to look at what he thought would be the purest substance on earth: rainwater. He was surprised that even this pure water contained live microorganisms (he called them animalcules) that he also observed in teeth scrapings. He drew pictures of what he saw and defined names for bacteria based on their shape. He called round bacteria cocci, rod shaped bacteria bacilli, and spiral bacteria spirochetes. He also described movement of bacteria by observing Giardia lamblia in stool specimens. He was the first person to record drawings of microbes and he introduced the world to the existence of microbes. The Royal Society of London published his drawings in their journal.

Robert Hooke (1685) He spent much of his life working with microscopes and improved their design and capabilities. He was the first to observe cells while looking at tree bark. He reported that living things were composed of little boxes that he called “cells.”

THE SPONTANEOUS GENERATION DEBATE

Francisco Redi (1668) He doubted the theory of spontaneous generation. He believed that maggots in meat were caused by flies laying eggs, not by spontaneous generation as commonly believed. He filled six jars with decaying meat. He noted that no maggots grew in the meat when the jars were covered. It was argued that air was needed for the vital force to create life. Therefore, he put a cheesecloth over half of the jars so that flies could not come in and lay their eggs, but air could get in. He demonstrated that maggots grew only in the meat

2 in which the flasks were not covered. However, the theory of spontaneous generation persisted because of the religious beliefs of the time.

John Needham (1749) He was a biologist and a priest who believed in spontaneous generation and he argued Redi’s theory. He boiled nutrient broth to kill all microbes, put a cork in the flask, then let it sit for a few days. He then took a culture of the broth and found that it contained microbes. Thus, he “proved” that life comes only from God and a “vital force”. He then had the scientists and the Church on his side. He did not know that the cork stopper was not sterilized, so microbes on it dropped into the broth.

For the next 30 years, various scientists who disagreed with spontaneous generation tried to disprove Needham’s experiment. One scientist said that Needham’s cork was contaminated, so he repeated the experiment except he sealed off the top of the glass by melting it; no microbes grew, so he said that spontaneous generation was false. His opponents said that the “vital force” that creates life is in the air, and that since he sealed off the air, the microbes could not spontaneously generate. Then other scientists would repeat the experiment with their own modifications. The debate over spontaneous generation raged on for 30 years, dividing the scientists from the religious clergy.

In the meantime, much of the world’s commerce was on agriculture, so there was a great concern for keeping cattle and sheep alive. The major diseases of this era were anthrax, the Black Plague, smallpox, and leprosy. There were also concerns about diseases of the money-making crops, including grapes, tobacco, and silkworms. While some scientists continued the debate over spontaneous generation, others turned their attention to the diseases at hand.

Louis Pasteur (1880) 150 years later, Pasteur joined the spontaneous generation debate. He made a glass flask with an “S” shaped bend in it so that bacteria could not enter into it. He placed chicken broth in the flask and boiled it so that it was sterile. Over the next few weeks, he observed that there was no bacterial growth in the broth, so this proved that spontaneous generation was impossible. No bacteria could grow unless pre-existing bacteria were able to get in to the nutrient broth. That ended the debate and the theory of spontaneous generation was rejected and the theory of biogenesis was accepted.

Edward Jenner, MD (1796) Jenner invented the first vaccine; it was for smallpox, the first disease to be eliminated from the earth by vaccine. Some of his patients, especially women who milked cows, would come in with lesions filled with pus on their hands. He went to their farms to examine the cattle and noticed lesions on the udders of the cattle as well.

Around this time smallpox was greatly feared, as one in three of those who contracted the disease died, and those who survived were commonly badly disfigured. Noting the common observation that milkmaids did not generally get smallpox, Jenner theorized that the pus in the blisters which milkmaids received from cowpox (a disease similar to

3 smallpox, but much less virulent) protected the milkmaids from smallpox. In 1796, Jenner tested his theory by inoculating James Phipps, a young boy, with material from the cowpox blisters of the hand of Sarah Nelmes, a milkmaid who had caught cowpox from a cow called Blossom. Phipps was the 17th case described in Jenner's first paper on vaccination.

Jenner inoculated Phipps with cowpox pus in both arms on one day. This produced a fever and some uneasiness but no great illness. Later, he injected Phipps with variolous material, (weakened smallpox). No disease followed. Jenner reported that later the boy was again challenged with variolacious material and again showed no sign of infection. Thus, he discovered that a genetically related microbe could produce immunity to the smallpox pathogen.

The word vaccination comes from the Latin vaccinia, cowpox, from vacca, cow. In 1980, the World Health Organization declared smallpox an eradicated disease.

Rudolf Virchow (1858) He helped create the Cell Theory (biogenesis theory), which said: 1. All living things are composed of cells. 2. Cells are the smallest working units of living things. 3. All cells come from preexisting cells by cell division.

Agostino Bassi (1844) He showed that a silkworm disease was caused by a fungus.

The Golden Age of Microbiology (1880-1930)

Louis Pasteur (1857-1914) Once the debate over spontaneous generation was over, the Golden Age of Microbiology began. Louis Pasteur went on to discover three vaccines; the first was by accident. During his time, chicken cholera was hurting the poultry industry. Pasteur made a pure culture of chicken cholera and told his assistant to inoculate a chicken. They both left on vacation and forgot to inoculate the chicken. When they came back, they inoculated the chicken with the same culture, which had run out of nutrients and had become weak. They found that the chicken never developed symptoms. They then tested all of the chickens to make sure that weakened culture did not cause symptoms in any chickens. They then exposed the chickens to chicken cholera and none of them became sick. They therefore discovered the concept of an immune system. They found that the old culture was weakened (he called this “attenuated”), and thus he discovered how to make the first vaccine for chicken cholera. Later, he found the causative agent for anthrax and invented the sheep anthrax vaccine. He also invented the rabies vaccine. A vaccine is a form or artificial immunity.

In 1854 Pasteur was appointed Dean and professor of chemistry at the Faculty of Sciences in Lille, France. He was told by the Minister of Public Instruction that science

4 for only science's sake was not appropriate. The Minister told the University Staff that "whilst keeping up with scientific theory, you should, in order to produce useful and far reaching results, appropriate to yourselves the special applications suitable to the real wants of the surrounding country." The town of Lille was an industrial town with a number of distilleries and factories.

Pasteur enjoyed taking his students on tours of the factories and was quick to advise the managers that he was available to help solve their problems. In the summer of 1856, M. Bigot, father of one of his students in chemistry, called upon Pasteur to help him overcome difficulties he was having manufacturing alcohol by fermentation of beetroot. Often, instead of alcohol, Bigot's fermentations yielded lactic acid.

In those days, fermentation leading to production of wine, beer and vinegar was believed to be a straightforward chemical breakdown of sugar to alcohol just by adding mild heat.

They did realize that yeast cells were in the fermenting vats of wine, and were recognized as being live organisms, but they were believed simply to be a product of fermentation.

Pasteur believed that yeast was the cause of, and not the product of, fermentation. The wine, beer and vinegar manufacturers were plagued by serious economic problems related to their fermentations. Yields of alcohol might suddenly fall off; wine might unexpectedly grow sour, vinegar might not be formed and lactic acid might appear in its place; the quality and taste of beer might unexpectedly change making quality control a nightmare! All too often the producers would be forced to throw out the resultant batches, start anew, and sadly have no better luck!

Into M. Bigot's factory, microscope in hand, came Pasteur. He quickly found three clues that allowed him to solve the puzzle of alcoholic fermentation. First, when alcohol was produced normally, the yeast cells were plump and budding. But when lactic acid would form instead of alcohol, small rod like microbes were always mixed with the yeast cells.

Second, analysis of the batches of alcohol showed that complex organic compounds were being formed during the fermentation. This indicated that some additional process other than sugar breaking down into alcohol must be involved. (He later called this process fermentation).

Third, Pasteur concluded and was able to prove that living cells, the yeast, were responsible for forming alcohol from sugar, and that contaminating microorganisms turned the fermentations sour! By showing the connection between spoilage of food and bacteria, is established the relationship between microbes and disease. This was critical in the development of the field of microbiology. Since he solved the problem of beer and wine spoilage, he saved the French wine industry.

One of Pasteur’s famous quotes is “Chance favors a prepared mind”. Many of the microbiology pioneers before and after Pasteur’s time would be examples of this quote.

5 Over the next several years Pasteur identified and isolated the specific microorganisms responsible for normal and abnormal fermentations in production of wine, beer, vinegar. He showed that if he heated wine, beer, milk to moderately high temperatures for a few minutes, he could kill living microorganism and thereby sterilize (pasteurize), the batches and prevent their degradation. If pure cultures of microbes and yeasts were added to sterile mashes uniform, predictable fermentations would follow. Pasteur used the term fermentation to describe the changes brought about by yeasts and other microorganisms to make alcohol products. He found that some microbes could live in anaerobic conditions (without oxygen) by fermenting sugars.

Pasteur also developed the concept of pasteurization. In this process milk is boiled at 58°C for 30 minutes to destroy microbes that spoil milk, beer, and wine. Shortly after that, the relationship between microbes, disease, immunity, and anti-microbial drugs was discovered.

Pasteur was the first to study diseases and develop the “Germ Theory of Disease”. He said that germs caused disease, not exposure to the elements, etc. This was useful in Streptococcus, Staphylococcus, pneumococcus, and anthrax infections.

Louis Pasteur went on to work with anthrax, silkworm disease, and rabies. He developed the first vaccine for rabies, which is a disease that spreads to the brain and spinal cord. Rabies is caused by a virus and is too small to see under a microscope. Therefore, he was the first to work with human diseases caused by viruses. He worked with rabbits that had rabies; he dissected out the spinal cords, put them in a jar, let them drive for a few weeks, ground them up into a powder, and made his suspension. He then inoculated dogs with this rabbit tissue. When the dogs were exposed to rabies, they did not get the disease.

A young boy named Joseph Meister was bitten by a rabid animal. Back then, everyone who was exposed to rabies died. Pasteur injected the boy’s abdomen with a vial of the spinal cord suspension once a week for 12 weeks. He was the first person to survive rabies. After Louis Pasteur died, Joseph became a caretaker of Pasteur’s estate. During World War II the Nazis invaded Paris. They wanted to raid Pasteur’s tomb, but rather than allow that, Joseph committed suicide.

Robert Koch, MD (1890) Unlike Pasteur, Koch was an MD, and he worked with a team of scientists, rather than alone, like Pasteur. He developed “Koch’s Postulates” which establish the cause of disease. This was developed using the scientific method, including stating a hypothesis and drawing a conclusion. He studied anthrax in sheep and cattle, and he also discovered the cause of tuberculosis. He is the one that proved the link between microbes and disease. He demonstrated that a specific microbe causes a specific disease.

Koch’s Postulates 1. Observe the same pathogen in every case of the disease. Then obtain the disease causing microbe from the sick animal via a sample. 2. Isolate this microbe in pure culture.

6 3. Inoculate a healthy animal with this pure culture, and the healthy animal should develop the same disease. 4. Re-isolate the microbe from the second animal. If it is the same microbe obtained from the first animal, this proves the etiology (cause) of the disease. For instance, Koch’s postulates were applied to the study of the AIDS virus, and by the 1990’s, all of them contributed to our understanding of the disease.

Koch is also the one who developed the use of agar, a solid medium used to make a pure culture of a certain organism. Colonies of bacteria from the patient’s specimen grew on the agar. After examining each colony under a microscope, he selected one that he thought was causing the disease. This one colony was then scraped off and placed on a new agar plate, creating a pure culture of the organism that he wanted to study. He was known for quoting Pasteur by saying “Chance favors a prepared mind”.

Joseph Lister (1827-1912) During his lifetime, 50% of people who had surgeries died from post-op infections. It was thought that exposure of a wound to oxygen caused the infection, but Lister doubted that explanation. He was put in charge of a new hospital in London that was designed to keep air away from post-op wounds. However, 50% of the amputation cases still were dying.

Then, in 1865, when Louis Pasteur suggested that decay was caused by living organisms in the air, Lister made the connection with wound sepsis. He considered that microbes in the air were likely causing the putrefaction and had to be destroyed before they entered the wound.

In the previous year Lister had heard that carbolic acid was being used to treat sewage in cattle fields that were causing a parasite disease in the cattle.

Lister began to clean wounds and dress them using a solution of carbolic acid. He reported that the surgery patients were now recovering without infections. In 1877 he performed the first surgery using aseptic conditions, including sterilization of instruments and hand washing before surgeries. Thus, he is the founder of aseptic medicine. Later, his product was refined by another doctor who named it Listerine® after Dr. Lister.

Ignaz Semmelwise (1847) Discovered that the incidence of puerperal fever could be drastically cut by use of hand washing standards in obstetrical clinics. Puerperal fever (or childbed fever) was common in mid-19th-century hospitals and often fatal, with mortality at 10%-35%. He also advocated that midwives should wash their hands when going from one obstetric patient to another to prevent transmission of puerperal fever.

Paul Ehrlich (1854 –1915) He coined the term "chemotherapy" and developed the first antibiotic drug in modern medicine. They called it the “magic bullet”; it was the cure for syphylis.

7 After his clinical education in Berlin in 1886 he received a call from Robert Koch to join the Institute for Infectious Diseases in Berlin (1891).

Ehrlich spent two years in Egypt, recovering from tuberculosis. Thereafter he worked on the development of the diphtheria serum. Ehrlich received the Nobel Prize for Medicine in 1908. In 1906 he discovered the structural formula of atoxyl, a chemical compound that had been shown to be able to treat sleeping sickness. Following this discovery, he tried to create a less toxic version of the medicine. In 1909, on his 606th attempt, he developed Salvarsan, an antibiotic effective against syphilis. This was the first antibiotic; his work was of epochal importance, stimulating research that led to the development of sulfa drugs, penicillin and other antibiotics.

Von Behring (1901): First to study how a toxin (Diptheria) can be neutralized by an antitoxin. He was the first person to receive a Nobel Prize in medicine.

Ross (1902) He discovered that mosquitoes transmitted malaria.

Metchnikoff (1908): Discovered that white blood cells phagocytize (eat) foreign particles as a fundamental part of the immune response. He studied mobile, amoeba-like cells (later known as white blood cells) in starfish. He thought they served as part of the defences of these organisms and, to test this idea, he stuck the starfish with small thorns from a tangerine tree which had been prepared as a Christmas tree for his children. Next morning he found the thorns surrounded by the mobile cells, and, knowing that, when inflammation occurs in animals which have a blood vascular system, leucocytes escape from their blood vessels, it occurred to him that these leucocytes might take up and digest bacteria that get into the body. This began the field of immunology.

Alexander Fleming (1881-1955) Fleming is the key person involved in the discovery of penicillin. One morning in the fall of 1928, Fleming noticed a culture plate displaying a colorful green mold. The culture plate was covered with staphylococci except in the vicinity of the mold, which was near the edge of the plate where there was a transparent zone of inhibition. The bacteria that were close to the mold had not grown. Since his mind was prepared by his previous studies, Fleming knew he might be on to something that kills bacteria. He photographed the plate and made it permanent by exposing it to formalin vapor that killed and fixed both the bacteria and the mold. (The original plate is now in the British Museum.) Fleming investigated a list of microorganisms that were inhibited by the mold; among them were streptococci, staphylococci, pneumococci and meningococci. Fleming identified the mold as a Penicillium that is similar some molds grown on bread. In 1945 Fleming received the Nobel Prize in Medicine and Physiology for his work with penicillin. NOTE: antibiotics are chemicals that kill bacteria. It is not the same thing as an antibody, which is a white blood cell’s natural protein that kills bacteria.

8 Chain and Florey The biggest problem was how to produce large quantities of penicillin, especially for casualties during WWII. American scientists Chain and Florey were successful in producing it for large-scale manufacture.

Rebecca Lancefield (1895 - 1981) American microbiologist. She is most famous for her serological classification of streptococcal bacteria based on the antigens in the cells walls.

She also demonstrated that one of these groups, group A streptococci (S. pyogenes), was specific to humans and human disease, including pharyngitis ("Strep throat"), scarlet fever, rheumatic fever, and impetigo. Group B streptococci were subsequently shown to be associated with neonatal disease.

Frederick Griffith (1928): found a “transformation principle” in bacteria, known as DNA today. Non-virulent streptococci (can’t cause disease) were formed into virulent strains.

Watson and Crick opened up the second golden age of microbiology when they discovered the structure of the DNA molecule in 1953. Watson and Crick were awarded the 1962 Nobel Prize award, for their body of research on nucleic acids. Their work became the basis for the Human Genome Project, which was a world-wide effort to list all of the sequences of amino acids in human DNA. This knowledge is helping to understand genetic disorders. This also opened up the field of Molecular Genetics, understanding how genes work at a molecular level.

François Jacob and Jacques Monod (1965), discovered the role of messenger RNA in protein synthesis. They won the Nobel Prize in Medicine in 1965.

Delbruck and Hershey (1969): Received the Nobel prize for their discoveries concerning the replication mechanism and the genetic structure of viruses.

Tonegawa (1987) Antibody genetics: To achieve the diversity of antibodies needed to protect against any type of antigen, the immune system would require millions of genes coding for different antibodies, if each antibody was encoded by one gene. Instead, as Tonegawa showed in a landmark series of experiments, genetic material can rearrange itself to form the vast array of available antibodies.

Prusiner (1997): American neurologist whose discovery of the disease-causing protein called prion in 1982 won him the 1997 Nobel Prize for Physiology or Medicine. Prions are simpler than viruses and can cause disease. They have no DNA or RNA. They cause a brain disease called spongiform encephalopathy (bovine form is Mad Cow Disease).

9 SELECTED NOBEL PRIZES IN PHYSIOLOGY 1901 Behring diphtheria antitoxin 1902 Ross malaria transmission 1905 Koch TB bacterium 1908 Metchnikoff phagocytosis 1945 Fleming, Chain, Florey penicillin 1969 Delbruck, Hershey viral replication 1987 Tohegawa antibody genetics 1997 Prusiner prions