The Dark-Field Microscope:-

Living, unstained cells and organisms can be observed by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image. The field surrounding a specimen appears black, while the object itself is brightly illuminated; because the background is dark, this type of microscopy is called dark-field microscopy. Considerable internal structure is often visible in larger eucaryotic microorganisms. The dark-field microscope is used to identify bacteria like the thin and distinctively shaped Treponema pallidum, the causative agent of syphilis.

The simplest way to convert a microscope to dark-field microscopy is to place 

(a) a dark-field stop underneath 

(b) the condenser lens system. 

The condenser then produces a hollow cone of light so that the only light entering the objective comes from the specimen.

Microscope Resolution:-
The most important part of the microscope is the objective, which must produce a clear image, not just a magnified one. Thus resolution is extremely important. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. Much of the optical theory underlying microscope design was developed by the German physicist Ernst Abbé in the 1870s. The minimum distance between two objects that reveals them as separate entities is given by the Abbé equation, in which lambda  is the wavelength of light used to illuminate the specimen and n sin theta is the numerical aperture (NA).

As d becomes smaller, the resolution increases, and finer detail can be discerned in a specimen. The preceding equation indicates that a major factor in resolution is the wavelength of light used. The wavelength must be shorter than the distance between two objects or they will not be seen clearly. Thus the greatest resolution is obtained with light of the shortest wavelength, light at the blue end of the visible spectrum.

The Light Microscope

Microbiologists currently employ a variety of light microscopes in their work; bright-field, dark-field, phase-contrast, and fluorescence microscopes are most commonly used. Modern microscopes are all compound microscopes. That is, the magnified image formed by the objective lens is further enlarged by one or more additional lenses.

The Bright-Field Microscope:-
The ordinary microscope is called a bright-field microscope because it forms a dark image against a brighter background. The microscope consists of a sturdy metal body or stand composed of a base and an arm to which the remaining parts are attached . A light source, either a mirror or an electric illuminator, is located in the base. Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image.

The stage is positioned about halfway up the arm and holds microscope slides by either simple slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide around smoothly during viewing by use of stage control knobs. The substage condenser is mounted within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in simpler microscopes but can be adjusted vertically in more advanced models.

The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes and are called binocular microscopes. The body assembly itself contains a series of mirrors and prisms so that the barrel holding
the eyepiece may be tilted for ease in viewing . The nosepiece holds three to five objectives with lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly. Ideally a microscope should be parfocal—that is, the image should remain in focus when objectives are changed.

The objective lens forms an enlarged real image within the microscope, and the eyepiece lens further magnifies this primary image. When one looks into a microscope, the enlarged specimen image, called the virtual image, appears to lie just beyond the stage about 25 cm away. The total magnification is calculated by multiplying the objective and eyepiece magnifications together.

The Future of Microbiology:-

As the preceding sections have shown, microbiology has had a profound influence on society. What of the future? Science writer Bernard Dixon is very optimistic about microbiology’s future for two reasons. First, microbiology has a clearer mission than do many other scientific disciplines. Second, it is confident of its
value because of its practical significance. Dixon notes that microbiology is required both to face the threat of new and reemerging human infectious diseases and to develop industrial technologies that are more efficient and environmentally friendly. What are some of the most promising areas for future microbiological research and their potential practical impacts? What kinds of challenges do microbiologists face? The following brief
list should give some idea of what the future may hold:

 New infectious diseases are continually arising and old diseases are once again becoming widespread and
destructive. AIDS, hemorrhagic fevers, and tuberculosis are excellent examples of new and reemerging infectious diseases. Microbiologists will have to respond to these threats, many of them presently unknown.

 Microbiologists must find ways to stop the spread of established infectious diseases. Increases in antibiotic
resistance will be a continuing problem, particularly the spread of multiple drug resistance that can render a
pathogen impervious to current medical treatment. Microbiologists have to create new drugs and find ways to
slow or prevent the spread of drug resistance. New vaccines must be developed to protect against diseases such as AIDS.

1. It will be necessary to use techniques in molecular biology and recombinant DNA technology to solve these problems. Research is needed on the association between infectious agents and chronic diseases such as autoimmune and cardiovascular diseases. It may be that some of these chronic afflictions partly result from infections. 
2. We are only now beginning to understand how pathogens interact with host cells and the ways in which diseases arise. There also is much to learn about how the host resists pathogen invasions. Microorganisms are increasingly important in industry and environmental control, and we must learn how to use them in a variety of new ways. For example, microorganisms can (a) serve as sources of high-quality food and other practical products such as enzymes for industrial applications, (b) degrade pollutants and toxic wastes, and (c) be used as vectors to treat diseases and enhance agricultural productivity. 
3. There also is a continuing need to protect food and crops from microbial damage.Microbial diversity is another area requiring considerable research. Indeed, it is estimated that less than 1% of the earth’s microbial population has been cultured. We must develop new isolation techniques and an adequate classification of microorganisms, one which includes those microbes that cannot be cultivated in the laboratory. Much work needs to be done on microorganisms living in extreme environments. The discovery of new microorganisms may well lead to further advances in industrial processes and enhanced environmental control. 
4.  Microbial communities often live in biofilms, and these biofilms are of profound importance in both medicine and microbial ecology. Research on biofilms is in its infancy; it will be many years before we more fully understand their nature and are able to use our knowledge in practical ways. In general, microbe-microbe interactions have not yet been extensively explored.
5. The genomes of many microorganisms already have been sequenced, and many more will be determined in the coming years. These sequences are ideal for learning how the genome is related to cell structure and what the minimum assortment of genes necessary for life is. Analysis of the genome and its activity will require continuing advances in the field of bioinformatics and the use of computers to investigate biological problems.
6. Further research on unusual microorganisms and microbial ecology will lead to a better understanding of the interactions between microorganisms and the inanimate world. Among other things, this understanding should enable us to more effectively control pollution. Similarly, it has become clear that microorganisms are essential partners with higher organisms in symbiotic relationships. Greater knowledge of symbiotic relationships can help improve our appreciation of the living world. It also will lead to improvements in the health of plants, livestock, and humans. 
7. Because of their relative simplicity, microorganisms are excellent subjects for the study of a variety of fundamental questions in biology. For example, how do complex cellular structures develop and how do cells communicate with one another and respond to the environment?
8. Finally, microbiologists will be challenged to carefully assess the implications of new discoveries and
   technological developments. They will need to communicate a balanced view of both the positive and
   negative long-term impacts of these events on society. The future of microbiology is bright.
9. The microbiologist René Dubos has summarized well the excitement and promise of microbiology: How extraordinary that, all over the world, microbiologists are now involved in activities as different as the study of gene structure, the control of disease, and the industrial processes based on the phenomenal ability of microorganisms to decompose and synthesize complex organic molecules. 
10. Microbiology is one of the most rewarding of professions because it gives its practitioners the opportunity to be in contact with all the other natural sciences and thus to contribute in many different ways to the betterment of human life.
    

Industrial Microbiology and Microbial Ecology:-

Although Theodore Schwann and others had proposed in 1837 that yeast cells were responsible for the conversion of sugars to alcohol, a process they called alcoholic fermentation, the leading chemists of the time believed microorganisms were not involved. They were convinced that fermentation was due to a chemical instability that degraded the sugars to alcohol. Pasteur did not agree. It appears that early in his career Pasteur became interested in fermentation because of his research on the stereochemistry of molecules. He believed that fermentations were carried out by living organisms and produced asymmetric products such as amyl alcohol that had optical activity.

There was an intimate connection between molecular asymmetry, optical activity, and life. Then in 1856 M. Bigo, an industrialist in Lille, France, where Pasteur worked, requested Pasteur’s assistance. His business produced ethanol from the fermentation of beet sugars, and the alcohol yields had recently declined and the product had become sour. Pasteur discovered that the fermentation was failing because the yeast normally responsible for alcohol formation had been replaced by microorganisms producing lactic acid rather than ethanol. In solving this practical problem, Pasteur demonstrated that all fermentations were due to the activities of specific yeasts and bacteria, and he published several papers on fermentation between 1857 and 1860.

His success led to a study of wine diseases and the development of pasteurization  to preserve wine during storage. Pasteur’s studies on fermentation continued for almost 20 years. One of his most important discoveries was that some fermentative microorganisms were anaerobic and could live only in the absence of oxygen, whereas others were able to live either aerobically or anaerobically. Fermentation ; The effect of oxygen on microorganisms.

A few of the early microbiologists chose to investigate the ecological role of microorganisms. In particular they studied microbial involvement in the carbon, nitrogen, and sulfur cycles taking place in soil and aquatic habitats. Two of the pioneers in this endeavor were Sergei N. Winogradsky (1856–1953) and Martinus
W. Beijerinck (1851–1931)

The Russian microbiologist Sergei N. Winogradsky made many contributions to soil microbiology. He discovered that soil bacteria could oxidize iron, sulfur, and ammonia to obtain energy, and that many bacteria could incorporate CO2 into organic matter much like photosynthetic organisms do. Winogradsky also
isolated anaerobic nitrogen-fixing soil bacteria and studied the decomposition of cellulose. Martinus W. Beijerinck was one of the great general microbiologists who made fundamental contributions to microbial
ecology and many other fields. He isolated the aerobic nitrogenfixing bacterium Azotobacter; a root nodule bacterium also capable of fixing nitrogen ; and sulfatereducing bacteria. Beijerinck and Winogradsky developed the enrichment-culture technique and the use of selective media , which have been of such great importance in microbiology.

Immunological Studies

In this period progress also was made in determining how animals resisted disease and in developing techniques for protecting humans and livestock against pathogens. During studies on chicken cholera, Pasteur and Roux discovered that incubating their cultures for long intervals between transfers would attenuate  the bacteria, which meant they had lost their ability to cause the disease. If the chickens were injected with these attenuated cultures, they remained healthy but developed the ability to resist the disease. He called the attenuated culture a vaccine in honor of Edward Jenner because, many years earlier, Jenner had used vaccination with material from co wpox lesions to protect people against smallpox .Shortly after this, Pasteur and Chamberland developed an attenuated anthrax vaccine in two ways: by treating cultures with potassium bichromate and by incubating the bacteria at 42 to 43°C.

Pasteur next prepared rabies vaccine by a different approach. The pathogen was attenuated by growing it in an abnormal host, the rabbit. After infected rabbits had died, their brains and spinal cords were removed and dried. During the course of these studies, Joseph Meister, a nine-year-old boy who had been bitten by a rabid
dog, was brought to Pasteur. Since the boy’s death was certain in the absence of treatment, Pasteur agreed to try vaccination. Joseph was injected 13 times over the next 10 days with increasingly virulent preparations of the attenuated virus. He survived. In gratitude for Pasteur’s development of vaccines, people from around the world contributed to the construction of the Pasteur Institute in Paris, France. One of the initial tasks of the Institute was vaccine production.

After the discovery that the diphtheria bacillus produced a toxin, Emil von Behring (1854–1917) and Shibasaburo Kitasato (1852–1931) injected inactivated toxin into rabbits, inducing them to produce an antitoxin, a substance in the blood that would inactivate the toxin and protect against the disease. A tetanus antitoxin was then prepared and both antitoxins were used in the treatment of people.

The antitoxin work provided evidence that immunity could result from soluble substances in the blood, now known to be antibodies . It became clear that blood cells were also important in immunity (cellular immunity) when Elie Metchnikoff (1845–1916) discovered that some blood leukocytes could engulf disease-causing bacteria . He called these cells phagocytes and the process phagocytosis.

Molecular Koch’s Postulates

Although the criteria that Koch developed for proving a causal relationship between and a microorganism and a specific disease have been of immense importance in medical microbiology, it is not always possible to apply them in studying human diseases. For example, some pathogens cannot be grown in pure culture outside the host; because other pathogens grow only in humans, their study would require experimentation on people. The identification, isolation, and cloning of genes responsible for pathogen virulence  have made possible a new molecular form of Koch’s postulates that resolves some of these difficulties. The emphasis is on the virulence genes present in the infectious agent rather than on the agent itself.

The molecular postulates can be briefly summarized as follows:

1. The virulence trait under study should be associated much more with pathogenic strains of the species than with nonpathogenic strains. Box 1.2 Molecular Koch’s Postulates

2. Inactivation of the gene or genes associated with the suspected virulence trait should substantially decrease pathogenicity.

3. Replacement of the mutated gene with the normal wild-type gene should fully restore pathogenicity.

4. The gene should be expressed at some point during the infection and disease process.

5. Antibodies or immune system cells directed against the gene products should protect the host. The molecular approach cannot always be applied because of problems such as the lack of an appropriate animal system. It also is difficult to employ the molecular postulates when the pathogen is not
well characterized genetically.

Eilshemius Hesse, the wife of Walther Hesse, one of Koch’s assistants . She suggested the use of agar as a solidifying agent—she had been using it successfully to make jellies for some time. Agar was not attacked by most bacteria and did not melt until reaching a temperature of 100°C. One of Koch’s assistants, Richard Petri, developed the petri dish , a container for solid culture media. These developments made possible the isolation of pure cultures that contained only one type of bacterium, and directly stimulated progress in all areas of bacteriology. Isolation of bacteria and pure culture techniques .

Koch also developed mediasuitable for growing bacteria isolated from the body. Because of their similarity to body fluids, meat extracts and protein digests were used as nutrient sources. The result was the development of nutrient broth and nutrient agar, media that are still in wide use today. By 1882 Koch had used these techniques to isolate the bacillus that caused tuberculosis. There followed a golden age of about 30 to 40 years in which most of the major bacterial pathogens were isolated

A Scientific method

      Although biologists employ a variety of approaches in conducting  research, microbiologists and other experimentally oriented biologists  often use the general approach known as the scientific method. They first gather observations of the process to be studied and then  develop a tentative hypothesis an educated guess  to explain the observations . This step often is inductive and creative because there is no detailed, automatic technique for generating hypotheses. Next they decide what information is required to test the hypothesis and collect this information through observation or carefully designed experiments.After the information has been collected, they decide whether the hypothesis has been supported or falsified. If it has failed to pass the test, the hypothesis is rejected, and a new explanation or hypothesis is constructed. Ifthe hypothesis passes the test, it is subjected to more severe testing. The procedure often is made more efficient by constructing and testing alternative  hypotheses and then refining the hypothesis that survives testing.This general approach is often called the hypothetico-deductive method.One deduces predictions from the currently accepted hypothesis and tests them. In deduction the conclusion about specific cases follows logically from a general premise. Induction is the opposite.A general conclusion is reached after considering many specific examples.
Both types of reasoning are used by scientists.When carrying out an experiment, it is essential to use a control  group as well as an experimental group. The control group is treated precisely the same as the experimental group except that the experimental manipulation is not performed on it. In this way one can be sure that any
changes in the experimental group are due to the experimental manipulation rather than to some other factor not taken into account.
If a hypothesis continues to survive testing, it may be accepted as a valid theory. A theory is a set of propositions and concepts that provides a reliable, systematic, and rigorous account of an aspect of nature. It is important to note that hypotheses and theories are never absolutely  proven. Scientists simply gain more and more confidence in their accuracy as they continue to survive testing, fit with new observations and experiments,and satisfactorily explain the observed phenomena.

•  The suspected microorganism must be isolated and grown in a pure culture.

• The same disease must result when the isolated  microorganism is inoculated into a healthy host.

•  The same microorganism must be isolated again from the diseased host.

Although Koch used the general approach described in the postulates during his anthrax studies, he did not outline them fully until his 1884 publication on the cause of tuberculosis .Koch’s proof that Bacillus anthracis caused anthrax was independently confirmed by Pasteur and his coworkers. They discovered that after burial of dead animals, anthrax spores survived and were brought to the surface by earthworms. Healthy animals  then ingested the spores and became ill.