Basic Microbiology and Immunology PMC 301 (Pharm D-Clinical) PDF

BASIC MICROBIOLOGY AND IMMUNOLOGY PMC 301(PHARM D-CLINICAL) Dr. Mohamed Abd El Gawad Lecture 2 Dr. Mohamed Abd El Gawad  Pasteur and the Defeat of Spontaneous Generation The mid-to late nineteenth century saw the science of mic...

BASIC MICROBIOLOGY AND IMMUNOLOGY PMC 301(PHARM D-CLINICAL) Dr. Mohamed Abd El Gawad Lecture 2 Dr. Mohamed Abd El Gawad  Pasteur and the Defeat of Spontaneous Generation The mid-to late nineteenth century saw the science of microbiology blossom. The concept of spontaneous generation was crushed and the science of pure culture microbiology emerged. Several scientific giants emerged in this era, and the first was the Frenchman Louis Pasteur (1822–1895), a contemporary of Cohn. Pasteur was trained as a chemist Fermentations At the invitation of a local industrialist who was having problems making alcohol by the fermentation of beets, Pasteur began a detailed study of the mechanism of the alcoholic fermentation, at that time thought to be a strictly chemical process. The yeast cells in the fermenting broth were thought to be a complex chemical substance and a result, rather than a catalyst, of the fermentation. Dr. Mohamed Abd El Gawad  Microscopic observations and other simple but rigorous experiments convinced Pasteur that the alcoholic fermentation was catalyzed by living yeast cells. Indeed, in Pasteur’s own words: “... fermentation is associated with the life and structural integrity of the cells and not with their death and decay.” From this foundation, Pasteur began a series of classic experiments on spontaneous generation, experiments that are forever linked to his name and to the science of microbiology. Spontaneous Generation: The concept of spontaneous generation had existed since biblical times ‫اﻷزﻣﻨﺔ اﻟﺘﻮراﺗﯿﺔ‬ The basic idea of spontaneous generation can easily be understood. For example, if food is allowed to stand for some time, it is putrefied. When the putrefied material is examined microscopically, it is found to be teeming with bacteria and perhaps even higher organisms such as maggots and worms. Dr. Mohamed Abd El Gawad  Where do these organisms that are not apparent in the fresh food come from? Some people said they developed from seeds or germs that entered the food from t h e air. Others said they arose spontaneously from nonliving materials, that is, spontaneous generation. Who was right? Keen insight was necessary to solve this controversy, and this was exactly the kind of problem that appealed to Louis Pasteur. Pasteur was a powerful opponent of spontaneous generation. Following his discoveries about fermentation. Pasteur showed that microorganisms closely resembling those observed in putrefying materials could be found in the air. Pasteur concluded that the organisms found in putrefying materials originated from microorganisms present in the air and on the surfaces of the containers that held the materials. He postulated that cells are constantly being deposited on all objects and that they grow when conditions are favorable. Furthermore, Pasteur reasoned that if food were treated in such a way as to destroy all living organisms contaminating it, that is, if it were rendered sterile and then protected from further contamination, it should not putrefy. Dr. Mohamed Abd El Gawad  Pasteur used heat to eliminate contaminants. Other workers had shown that when a nutrient solution was sealed in a glass flask and heated to boiling for several minutes, it did not support microbial growth (of course, only if endospores were not present; see discussion of Cohn). Killing all the bacteria or other microorganisms in or on objects is a process we now call sterilization. Proponents of spontaneous generation criticized such experiments by declaring that “fresh air” was necessary for the phenomenon to occur. Boiling, so they claimed, in some way affected the air in the sealed flask so that it could no longer support spontaneous generation. In 1864 Pasteur countered this objection simply and brilliantly by constructing a swan-necked flask, now called a Pasteur flask (Figure 5). In such a flask nutrient solutions could be heated to boiling and sterilized. However, after the flask was cooled, the air was allowed to reenter, but bends in the neck (the “swan neck” design) prevented particulate matter (containing microorganisms) from entering the main body of the flask and causing putrefaction. Dr. Mohamed Abd El Gawad Figure 5. The defeat of spontaneous generation: Pasteur’s experiment with the swan-necked flask. (a) Sterilizing the contents of the flask. (b) If the flask remained upright, no microbial growth occurred. (c) If microorganisms trapped in the neck reached the sterile liquid, microbial growth ensued. Dr. Mohamed Abd El Gawad  Broth sterilized in a Pasteur flask did not putrefy, and microorganisms never appeared in the flask as long as the neck did not contact the sterile liquid. If, however, the flask was tipped to allow the sterile liquid to contact the contaminated neck of the flask (Figure 5c), putrefaction occurred and the liquid soon teemed with microorganisms. This simple experiment effectively settled the controversy surrounding spontaneous generation, and the science of microbiology was able to move ahead on firm footing. Incidentally, Pasteur’s work also led to the development of effective sterilization procedures that were eventually refined and carried over into both basic and applied microbiological research. Food science also owes a debt to Pasteur, as his principles are applied today in the canning and preservation of milk and other foods (pasteurization). Dr. Mohamed Abd El Gawad Other Accomplishments of Louis Pasteur: Pasteur went on to many other triumphs in microbiology and medicine beyond his seminal work on spontaneous generation. Some highlights include his development of vaccines for the diseases anthrax, fowl cholera, and rabies during a very scientifically productive period in his life from 1880 to 1890. Pasteur’s work on rabies was his most famous success, culminating in July of 1885 with the first administration of a rabies vaccine to a human, a young French boy named Joseph Meister who had been bitten by a rabid dog. In those days, a bite from a rabid animal was akin to a death sentence. Dr. Mohamed Abd El Gawad  News of the success of Meister’s vaccination, and that of a young shepherd boy, Jean Baptiste Jupille, administered shortly thereafter, spread quickly, and within a year nearly 2500 people had come to Paris to be treated with Pasteur’s rabies vaccine. In Conclusion  Louis Pasteur is best remembered for his ingenious experiments showing that living organisms were not spontaneously generated from nonliving matter. Pasteur’s work in this area led to many of the basic techniques central to the science of microbiology, including the concept and practice of sterilization. Robert Koch (1843–1910), Infectious Disease, and the Rise of Pure Culture Microbiology: Proof that microorganisms could cause disease provided perhaps the greatest impetus for the development of the science of microbiology. Even in the sixteenth century it was thought that something that induced a disease could be transmitted from a diseased person to a healthy person. Dr. Mohamed Abd El Gawad  After the discovery of microorganisms, it was widely believed that they were responsible, but definitive proof was lacking. The Germ Theory of Disease and Koch’s Postulates: In his early work Koch studied anthrax, a disease of cattle and occasionally of humans. Anthrax is caused by an endospore-forming bacterium called Bacillus anthracis. By careful microscopy and by using special stains, Koch established that the bacteria were always present in the blood of an animal that was succumbing to the disease. However, Koch reasoned that mere association of the bacterium with the disease was not proof that it actually caused the disease. Instead, the bacterium might be a result of the disease. How could cause and effect be linked? With anthrax Koch sensed an opportunity to study cause and effect experimentally, and his results formed the standard by which infectious diseases have been studied ever since. Dr. Mohamed Abd El Gawad  Koch used mice as experimental animals. Using all of the proper controls, Koch demonstrated that when a small amount of blood from a diseased mouse was injected into a healthy mouse, the latter quickly developed anthrax. He took blood from this second animal, injected it into another, and again obtained the characteristic disease symptoms. However, Koch carried this experiment a critically important step further. He discovered that the anthrax bacteria could be grown in nutrient fluids outside the animal body and that even after many transfers in laboratory culture, the bacteria still caused the disease when inoculated into a healthy animal.  On the basis of these and related experiments carried out in his seminal work on the causative agent of tuberculosis, Koch formulated a set of rigorous criteria, now known as Koch’s postulates, for definitively linking a specific microorganism to a specific disease: Dr. Mohamed Abd El Gawad 1. The disease-causing organism must always be present in animals suffering from the disease and should not be present in healthy animals. 2. The organism must be cultivated in a pure culture away from the animal body 3. The isolated organism must cause the disease when inoculated into a healthy susceptible animal 4. The organism must be reisolated from these experimental animals and cultured again in the laboratory, after which it should still be the same as the original organism Koch’s postulates are summarized in Figure 6. Koch’s postulates were a monumental step forward in the study of infectious diseases. The postulates not only offered a means for linking the cause and effect of an infectious disease, but also stressed the importance of laboratory culture of the putative infectious agent. With these postulates as a guide, Koch, his students, and those that followed them discovered the causative agents of most of the important infectious diseases of humans and other animals. Dr. Mohamed Abd El Gawad  These discoveries led to the development of successful treatments for the prevention and cure of many of these diseases, thereby greatly improving the scientific basis of clinical medicine and human health and welfare. Figure 6 Koch’s postulates for proving that a specific microorganism causes a specific disease. Note that following isolation of a pure culture of the suspected pathogen, laboratory culture of the organism should both initiate the disease and be recovered from the diseased animal. Establishing the correct conditions for growing the pathogen is essential, otherwise it will be missed. Dr. Mohamed Abd El Gawad