Historical Overview

Lecture 01 [Notes] Implications and Uses
Microbiology deals with microscopic organisms and structures: their morphology (structure), physiology (function), behavior and uses.

This study may include bacteria, fungi, mycoplasmas, some algae and protozoa, and the viruses. Viruses are acellular and are not considered living organisms. Rather, they are seen as obligate, intracellular parasites that require the machinery of a live host cell in order to replicate. As we shall see this definition can cause problems in the area of sterilization.

There are often separate courses provided to cover the vast array of organisms which are mentioned in microbiology: e.g. Mycology (the study of fungi), Protozoology (the study of protozoans - one celled animal-like organisms), Parasitology (the study of parasites from bacteria to protozoans to worms and flukes),and Virology (the study of viruses).

An introductory general course in microbiology will encompass a variety of topics including: bacteriology, immunology, epidemiology, toxicology, pharmacology - to mention but a few.
In addition, an understanding of microbial and eukaryotic bio-chemistry, cytology, ecology, physiology and taxonomy will also be included.

Microorganisms play many roles within the biosphere:
within ecosystems they serve as:
decomposers - mechanical breakdown, decay and dismemberment
transformers - reducing compounds to elemental form for use in various biogeochemical cycles, e.g. nitrogen cycle, Rhizobium
selective agents - weeding out the weak and infirm, keeping population size in check through disease production (except for the human population thanks to health care)
disease producers - plants, animals and humans with implications and costs

within medicine, agriculture, and industry:
they are involved in antibiotic production; water and waste contamination and purification; food spoilage, storage, and preservation; poisoning; dairy products; fertilizers, flavors in cheeses; fermentation of bread, brewing of wine,beer, ethyl alcohol; genetic engineering (gene splicing and expression as in antibiotic and hormone production)

Microbes are in us, on us, and all around us.
Do you know where Helicobacter pylori is found in your body?

They ruled this planet for at least 3 billion years before the evolution of eukaryotic life forms. Their descendants are within our cells as the mitochondria and in plants as the chloroplasts.
"Adam had 'em"

A brief historical look at various scientific and unscientific developments will show how Microbiology emerged and continues to grow as a vital and important field.
Many of the individuals and groups that influenced medicine and microbiology are often unknown or underrepresented. The article "How Islam Changed Medicine" is a case in point. Most people are unaware of early Arab contributions.

1670's Anton von Leeuwenhoek first to see microorganisms and record the information; verified by Royal Society
1830 Joseph J. Lister the father of Joseph (Lord) Lister published his work on improving the quality of microscopes - a major advance in the use of the microscope for visualization of microbes
1877 Robert Koch develops methods for staining and photographing bacteria
1878 Ernst Abbe describes oil immersion lens
1881 Paul Ehrlich introduces dye methylene blue into bacteriological staining
1881 Hans Christian Gram describes a new method for staining bacteria - the Gram stain (a differential stain)
1881 Robert Koch publishes his methods for the isolation of pure cultures by the use of semi-solid media, firmly establishing bacterio- logy as a science
1887 R.J. Petri describes new type of culture dish for semi-solid media
1911 Oskar Heimstadt invents fluorescent microscope
1932 Max Knoll &Ernst Ruska publish the first description of the transmission electron microscope (TEM)
1935 Frits Zernike publishes first description of the phase-contrast microscope

As new techniques for detection, characterization, and growth of microorganisms became available, it was inevitable that new microbes related to disease production would be discovered.

1877 Robert Koch Bacillus anthracis (anthrax)
1879 Albert Neisser Neisseria gonorrhea
1881 Alexander Ogston Staphylococcus aureus (pyogenic infections)
1882 Carl Gessard Pseudomonas aeruginosa (various)
1882 Robert Koch Mycobacterium tuberculosis
1882 Frederick Fehleisen Streptococcus pyogenes
1883 Theodor Klebs Corynebacterium diphtheriae
1884 Friedrich Loeffler Corynebacterium diphtheriae
1884 Arthur Nicolaier Clostridium tetani (anaerobe)
1884 Robert Koch Vibrio cholerae
1884 George Gaffky Salmonella typhi
1885 Gustav Hauser Proteus vulgaris (various)
1885 Theodor Escherich Escherichia coli (normal flora)
1886 Daniel Salmon & Theobald Smith Salmonella cholerae-suis (swine plague)
1887 David Bruce Brucella melitensis (brucellosis)
1888 August Gaertner Salmonella enteritidis (food poisoning)
1889 Shibasaburo Kitasato Clostridium tetani
1892 William Welch & George Nuttall Clostridium perfringens (gas gangrene)
1894 Alexandre Yersin Yersinia Pasteurella pestis (bubonic plague)
1897 Emile van Ermengem Clostridium botulinum
1898 Kiyoshi Shiga Shigella flexneri (bacterial dysentery)
1905 Fritz Schaudinn & Erich Haffman Treponema pallidum (syphilis)
1906 Jules Bordet & Octave Gengou Bordetella pertussis (whooping cough)
1909 Howard T. Ricketts Rickettsia rickettsii (Spotted Fever)
1912 Hideyo Noguchi Spirochaeta refringens (first pure culture of a spirochete)
1912 George W. McCoy & Charles W. Chapin Pasteurella tularensis (rabbit fever) (rediscovered and renamed Franciscella tularensis in the 1940's)

As more disease producing organisms were discovered, new insights into how the body fought these organisms were being developed. The following is a historical perspective in the area of immunology.

The basic idea of immunology was known to the early Greeks and recorded by the historian Theophrastus as he observed soldiers dying or recovering from both wounds and other infections.

The term "immune" was first applied to those soldiers who survived the Black Death (bubonic plague) and were therefore immune from battle field service. It was their chore to cart off and burn the dead bodies of plague victims. A tough way to start as the first male nurses.

1660's William Harvey discovers that blood circulates in a closed system of arteries, capillaries, and veins - thus disproving the early ideas of the Roman physician Galen
1798 Edward Jenner, an English country doctor, develops a vaccine for smallpox and initiates attenuation studies
1882 Pasteur, using some of Jenner's ideas, attenuates the agent responsible for fowl cholera and goes on to develop vaccines for anthrax and rabies

This work represents the beginning of what is called active immunization.
1884 Metchnikoff, a Russian scientist, describes "phagocytosis" by white blood cells (neutrophils); this work establishes a cellular aspect of immunology; Metchnikoff joins Pasteur
1884 Nuttal demonstrates the bactericidal action of serum: Bordet who later discovers the causative agent of whooping cough, explains the serum's action is due to special serum proteins called "antibodies"and other proteins with enzyme-like qualities called complement
1888 Roux and Yersin, both of whom work with Pasteur, isolate and describe the toxin produced by the diphtheria microbe
1890 von Behring and Kitasato isolate and describe the toxin produced by the tetanus organism

This work sets the stage for the development of passive immunization techniques.
1906 Wasserman adapts the "complement-fixation" reaction for use in syphilis testing
1930 Frobisher and Davis use the complement-fixation test for yellow fever testing

Since the 1960's the amount of information has increased dramatically. It has been suggested that during the 1980's the amount of information about immunology doubled every 2-3 years. Probably a lot sooner since the advent of computer databases.

The initial development of microbiology would not have been possible without the ability to see the microbes. From the time of Leeuwenhoek and earlier, crude microscopes were available. In the 20th and 21th centuries the types of microscopes that have been and will be developed has changed dramatically. These new microscopes now have the capability to see individual molecules and even individual atoms.
Labelled Photograph of Compound Brightfield Microscope

Some Additional Types of Microscopy - A Brief Review

The initial development of microbiology would not have been possible without the ability to see the microbes. From the time of Leeuwenhoek and earlier, crude microscopes were available. In the 20th and 21th centuries the types of microscopes that have been and will be developed has changed dramatically. These new microscopes now have the capability to see individual molecules and even individual atoms. Visit this site to see a comparison of standard light, dark field and phase contrast microscopy.

Microscopes Help Scientists Explore Hidden Worlds. This web site, located on the Nobel site, includes succinct information on microscopy, as well as interative simulators. This site provides an excellent model for what is possible on the web. The simulator requires the latest Shockwave Player (free from Macromedia at http://www.macromedia.com/), portraying phase contrast and transmission electron microscopy with surprising accuracy. Single page summaries are available on the history of microscopy, resolution limits, and four types of microscopy: phase contrast, fluorescence, transmission electron and scanning tunneling microscopy. This site is supported by Zeiss. (****) -S

Darkfield - look for picture of Buccal (Mouth) Epithelium
The darkfield microscope uses a modified CONDENSER so that light is prevented from passing directly through the specimen. The light is directed at the specimen from the sides and thus the only light seen is that which is scattered from the cells. This is similar to moonlight. This type of scope makes possible the observation, in the living state, of particles and cells so tiny that they are invisible in a conventional brightfield microscope. The Negative staining technique gives a similar result. This scope might be used for viewing the syphilis organism, Treponema pallidum, a small spiral microbe.

The fluorescent microscope makes use of the fact that certain chemicals and dyes give off light of one color when they are subjected to light of another color (wavelength). A filter in the light source absorbs the fluorescent light so that only the light coming from the organism or molecule comes through to be seen. The dye auromine has been used for TB organisms and gives a yellow light at a wavelength of 600 nm. Fluorescent staining is sometimes done to detect antibodies against syphilis and for detection of the rabies virus. One limitation is that the work must be done in a completely darkened area. A great deal of work in recent times has used green fluorescent protein (GFP) from a jellyfish and luciferin from the firefly for identifying molecules and parts of eukaryotic cells. The photomicrographs of human cheek cells show the use of GFP. It is also being used for extraction and separation of molecules.

Note the bacteria on the surface of this white blood cell.
*Used with permission from Purdue Cytometry.
Note bacteria on surface of cheek cell shown with fluorescent dyes

The phase-contrast microscope uses a modified DIAPHRAGM to place light out of phase. No staining is required and this scope allows visualization of living, unstained organisms. It is often used with wet mounts and hanging drop slides. It will show motility of organisms and the internal cellular structures will appear as darker, contrasting structures.

Transmission Electron Microscope (TEM) Visit the site and note the pictures.
Instead of a visible light source the TEM uses an electrical current to heat a tungsten filament to 20000 C, causing clouds of electrons to boil off around the wire. An Electron gun accelerates the electrons through the vacuum chamber of the scope. Streams of electrons pass through two (2) electromagnets known as CONDENSER LENS. This lens shapes the electron beam so that it passes through the material to be magnified. The material to be examined is mounted on a fine copper wire mesh. The electron beam passes through the material and enters two (2) more sets of electromagnets: the OBJECTIVE LENS and the PROJECTOR LENS. Both of these bend the electron beam and produce high magnification. The extremely short wavelengths of the electron beam are not visible. However, they strike a phosphorus-coated plate, enclosed in a leaded glass for safety. The plate glows and produces a visible image. This is the principle of the picture on a TV picture tube. The operator uses the image on the view plate to position materials and focus the electron beams. He usually exposes a photographic negative from which a photographic print can be made for study. The amount of magnification is determined by the electromagnets used. Magnifications range from 1,400X to 200,000X. The negative can be enlarged several times without loss of resolution producing a final magnification of over 1,000,000X. The major limitations of this technique include: long preparation time of the specimen; a need for very thin slices of the specimen; the use of the vacuum tube means materials must be dehydrated and thus no living materials can be studied.

Scanning Electron Microscope (SEM)Visit the site and note the pictures.
This scope was developed as an offshoot of work done during the space program to land a man on the moon. See how a SEM works at this site. Electrons do not pass through the specimen, but scan back and forth across it, allowing a view of the specimen surface in 3-D detail. The electron beam projected from a filament is focused into a fine pencil line by two (2) electromagnetic condenser lens and concentrated on the specimen which is held in a tilted mount. The current from the scanning generator causes this beam to scan back and forth across the specimen while at the same time causing a light spot to sweep across a cathode ray tube where the image will be seen. At this stage the tube looks similar to a TV screen just before the picture comes on. Then the beams that are scanning the specimen are caught by a signal detector and passed through a video amplifier the increases their volume. The signal produced modulates the brightness of the spot moving across the tube and the result is a TV-like image up to 50,000X specimen size. This same principle is used in getting pictures from the moon and other planets.

Scanning Tunneling Microscope (STM)
Since the STM was developed in 1981, it has made a dramatic impact on the understanding of surface structures of molecules. It can magnify molecules several billion times. Most biological molecules are too small to be seen with the light microscope and the electron beam of electron microscopes often damages their structure. The STM produces 3-D images of atoms and molecules without the use of destructive electron beams. The microscope works by using a procedure called electron tunneling. A sample must conduct electrons if it is to be imaged with the STM. The samples are coated with metals and the STM detects electrons jumping or "tunneling" from the surface of the specimen. An electrically conductive, needlelike probe, usually made of tungsten, scans the surface billionths of a millimeter above the sample. It follows the sample's surface outline by maintaining a constant distance from the surface. The probe is attached to a computer that projects a 3-D image onto a fluorescent screen. A team of scientists in California recently used the STM to obtain the first direct image of DNA. These new images show the three-dimensional structure of DNA and provide support for theories that DNA exists in several helical variations.

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