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Robert Koch, pictured in Figure 1-4, searched for the causes of many diseases. Through these investigations he and his laboratory developed many classic microbial techniques. He used adaptations of the staining methods of Carl Weigert to begin the process of distinguishing microbes and identifying pathogens. His lab was the first to isolate a disease-causing organism.
Figure 1.4 Robert Koch. The German Physician Robert Koch. Drawing by Tammi Henke
A major contribution to bacterial techniques was the development of methods using solid medium. For example LB medium or minimal medium for the cultivation of bacteria. Koch was convinced that microbes caused some diseases. However, to test this idea, he needed to isolate the causative agent. Almost all samples from diseased animals or any natural surface contained many different microbes and it was impossible to tell which one was the problem. A method was needed to separate these different bacteria. The most common method of isolation was to continually dilute a sample in liquid broth in hopes that at high enough dilution, only one type of microbe would be found. A problem with this method is that only the most populous microbe would be isolated, but that might not be one causing the disease. There were other technical problems as well with such a liquid-based system, so a solid medium would seem to provide distinct advantages. Koch had tried gelatin for these experiments with unsatisfactory results. Building on the work of Brefeld and Schroeter, Koch used potato slices as a solid medium and observed that a boiled potato left in the open air would develop tiny circular raised spots.
Examination of these spots revealed they were made up of microorganisms and each spot had just one type of microbe in it. He realized that these colonies were pure cultures of bacteria and probably arose from a single species of microbe from the air that landed on the potato. By boiling a potato, slicing it with a hot knife and keeping it in a sterile container with a lid, Koch could keep the potato sterile. But if a sample from a disease animal was smeared across the potato, colonies arose, each being pure isolates from the animal. By then testing these isolates in animals, Koch was able to isolate the cause of anthrax, Bacillus anthracis.
Potatoes failed to support the growth of many microorganisms and Koch and his laboratory were constantly frustrated by the lack of a good solid medium. Walter Hesse joined Koch's laboratory to do studies on air quality, showing a remarkable attention to detail and patience in his work. His wife, Angelina Fannie Hesse along with raising their three sons, also would assist her husband with his research in the laboratory. Walter was attempting to do his air quality experiments using medium containing gelatin as the solidifying agent. In the summertime, temperatures would often rise above the melting point of gelatin. In addition, microbes would often grow in the cultures that were capable of degrading gelatin and in both cases this would cause liquefaction of the medium, ruining the experiments. One day while eating lunch, the frustrated scientist asked Lina (as she was called) why her jellies and puddings stayed solid even in the hot summer temperatures. She told him about agar-agar, a heat resistant gelling agent that she had learned about while growing up in New York from a Dutch neighbor who had emigrated from Java.
Development of the new agent by Angelina and Walter led to a resounding success. Few microbes are able to degrade agar and it melts at 100 °C yet remains molten at temperatures above 45 °C. This allows the mixing of the agar with heat-sensitive nutrients and microbes. After solidification, it does not melt until a temperature of 100 °C is again attained, facilitating the easy cultivation of pathogens. It can also be stored for long periods of time, allowing the cultivation of slow-growing microorganisms. Any type of broth can be mixed with agar, giving great flexibility in the kinds of medium that can be made. Thus, many more types of microbes could be cultivated.
Koch's laboratory also developed methods of pure culture maintenance and aseptic technique. Aseptic technique involves the manipulation of pure cultures in a manner that prevents their contamination by outside microorganisms. Equally important, aseptic technique prevents their spread into the environment. Remember that Koch was studying some of the most devastating microbial pathogens of the period, and their release could potentially cause disease in the scientists working on them. These procedures were also absolutely critical because they allowed careful study of pure microorganisms, making it possible to identify the role of each microbe in a given situation.
Another problem in the cultivation of microbes was solved by Julius Petri while working in Koch's laboratory. Solid medium was poured on glass plates and allowed to spread and harden. Once cooled it allowed a solid surface for streaking. However, creation of these plates required great care since exposure to the air (and the microbes in it) often lead to contamination. In addition, to prevent contamination of plates during incubation, a cumbersome bell jar was used. If one wanted to view samples, the plate had to be removed from the jar, further exposing it to unwanted microbes in the air. In 1887 Petri developed shallow glass dishes, with one having a slightly larger diameter than the other. Medium is poured into the smaller dish and the larger one serves as a cover. This simple device solved all of the above problems and took on the name of its inventor, the petri plate.
These same techniques are essential in studying all microorganisms. Collectively the above techniques have been used to isolate and identify thousands of different microorganisms. As a testament to the significance of their achievement, these techniques are practiced with remarkably little change in every laboratory that works with microorganisms today. Figure 1-27 lists some of the early advances that helped to develop the practice of microbiology.
|1664||Robert Hooke is the first to use a microscope to describe the fruiting structures of molds. He also coined the term cell when using a microscope to look at cork, as the dead plant material in cork reminded him of a jail cell.|
|1673||Anton van Leeuwenhoek, a Dutch tradesman and skilled lens maker, is the first to describe microbes in detail.|
|1872||Ferdinand Julius Cohn publishes landmark paper on bacteria and the cycling of elements. In it is an early classification scheme that uses the name Bacillus.|
|1872||Oscar Brefeld reports the growth of fungal colonies from single spores on gelatin and the German botanist Joseph Schroeter grows pigmented bacterial colonies on slices of potato.|
|1877||Robert Koch develops methods for staining bacteria, photographing, and preparing permanent visual records on slides.|
|1881||Koch develops solid culture media and the methods for obtaining pure cultures of bacteria.|
|1882||Angelina Fannie and Walther Hesse in Koch's laboratory develop the use of agar as a support medium for solid culture.|
|1884||Hans Christian Gram develops a dye system for identifying bacteria [the Gram stain].|
|1887||First report of the petri plate by Julius R. Petri.|
|1915||M. H. McCrady establishes a quantitative approach for analyzing water samples using the most probable number, multiple-tube fermentation test.|
Figure 1.27 The development of early techniques in microbiology..