Introduction to Microbiology


     Microbiology is the branch of science that concerns itself with the study of organisms which are barely visible or invisible to the naked eye.  It is a central science in that it enhances our ability to understand the smallest of all biological entities and from this understanding build new knowledge about all living things.  Microbiology has a firm basis both in the pure and applied aspects of science, in that the knowledge gained from basic research into the biology of microorganisms can be applied to such widely varying areas as agriculture, manufacturing, the food industry, medicine, and genetic engineering.
 

Historical Overview

 

The Quest to Disprove the Theory of Spontaneous Generation

     Fundamentally curious by nature,  man has always been fascinated by the world of the unseen.  Unfortunately man is not always quick to look for logical explanations to phenomena which occur in nature, especially when those phenomena are not directly visible to the naked eye.  Sometimes, it is much simpler to ascribe such unseen happenings to supernatural sources rather than to look for rational explanations based upon observation, technique, and previous knowledge about the natural world.  Thus it is not surprising  to learn that throughout most of recorded history people strongly believed that life could arise from a non-living source (spontaneous generation), and that illness, disease, and decay were the results of curses, the breathing in of bad air, or the influence of bad blood.  That such beliefs persisted until the middle of the nineteenth century seems astonishing to us today, but it is wise to be mindful of the adage that "old beliefs die hard".

     One of the earliest attempts to dispel the belief in abiogenesis (spontaneous generation) was provided by the naturalist Francesco Redi (1621-1697).  Redi was a very methodical, observant researcher who sought to disprove the idea that flies spontaneously arise from rotting meat.  In his 1668 manuscript, Espererienze Intorno alla Generazione Degli Insetti (Observations on the Generation of Insects, 1668), Redi wrote that he:

         " began to believe that all worms found in meat were derived from flies, not from
           putrefaction.  I was confirmed by observing that, before the meat became wormy,
           there hovered over it flies of that very kind that later bred in it.  Belief unconfirmed
           by experiment is in vain."*
 
 

     To prove that what he observed was correct, Redi placed meat in several dishes, covering half of these with gauze.  He also checked his results with several empty dishes which served as controls.  He found that after several days, the dishes which contained uncovered meat were covered with maggots, but neither the covered meat, nor the empty dishes had similar infestations.  Though these results were incontrovertible, Redi used no means of close inspection to confirm them, so his work failed to convince those who relied on microscopic evidence.

     John Turberville Needham (1713-1781) was an English catholic priest who repeated Redi's experiment, but used boiled mutton broth sealed in corked containers to exclude exterior air.  While the idea behind this experiment was to kill microbes already present in the broth and prevent the entry of other agents of decay,  when the containers were opened, they were found to be full of microorganisms.  After repeating the experiment with several other broths of various kinds and getting the same results, Needham concluded that spontaneous generation actually did occur.  However, this conclusion was challenged by the Italian investigator Lazzaro Spallanzani (1729-1799), who assumed that boiling alone was not sufficient to destroy microbes unless performed for a sufficiently long period of time in the absence of air.  By setting up a series of open and closed broth treatments which were boiled for increasingly longer periods of time, Spallanzani was able to show that his initial assumption was correct.

     The experiments performed by Needham and Spallanzani were repeated by many other nineteenth-century researchers with varying results.  The major flaw inherent to all of these experiments was that they were designed to disprove, rather than to prove, thus any questionable result would bring the debate to life once more.  Some felt that the exclusion of air was the important factor which was responsible for the lack of growth of microorganisms in sealed broths, and this led to the belief that there must be some "active principle" present in air which allowed spontaneous generation to occur.  The debate would continue until 1861 with the publication of Mémoire sur les corpuscles organisés qui existent dans l'atmosphere by the great French researcher Louis Pasteur (1822-1895).  Pasteur was a physical chemist by training, but he pursued many research interests and was able to correctly demonstrate how milk sours (1857), what causes diseases and defects in wine (1866), and the cause of disease in silkworms (1862).  By 1859, Pasteur was convinced that microbes were the agents of decay and were responsible for many diseases.  He also concluded that microbes which cause broths and foodstuffs to go bad existed in air, but as separate entities from air, thus they could be excluded in such a way as to prevent decay.  To prove this, he performed a simple experiment, wherein boiled broths were placed in flasks which had necks bent into S-shaped curves.  Pasteur reasoned that if the microbes were indeed separate from air, they must be carried on particles of matter such as dust.  Since dust particles are heavier than air, they became trapped in the S-shaped portion of the flasks, thereby preventing bacteria from getting to the broths.  Fluids kept in such containers could be stored for long periods of time without fear of contamination.  To prove that his assumptions were correct, Pasteur simply broke the necks of the flasks, allowing dust to settle in the broths, which then became cloudy with the growth of microorganisms.  This settled the dispute over the presence of an "active principle" in air, since the broths were always exposed to air.

     Associated with these findings was the work of the English experimenter John Tyndall (1820-1893), who realized that some bacteria had the ability to form resistant structures called spores.  Tyndall demonstrated that previous researchers such as Needham had failed to realize that the spores of microbes could resist single boiling processes and give rise to new vegetative cells.  Through a series of boiling and cooling steps which first allowed spores to germinate and then killed the new cells which arose from them, Tyndall was able to produce truly sterile broths which remained pure for very long periods of time.  This technique was given the name "tyndallization" in his honor.  In light of the combined efforts of Pasteur and Tyndall, the debate over the reality of spontaneous generation was finally put to rest.
 

Early Microscopy

     The use of lenses and lens systems to increase the apparent size of an object is probably the most significant breakthrough in the development of microbiology as a true science.  One of the earliest individuals to realize the potential of lenses was Roger Bacon (1214-1294), a Franciscan who taught at Paris and Oxford.  Bacon understood the principle of refraction as it applies to curved lenses, and developed an accurate description of the way light is bent through a "burning glass" (magnifying lens).  He also made mention of the use of lenses in spectacles, and hinted at the use of multiple lens systems in optical instruments.  Later, Willibrord Snell (1591-1626) would develop the mathematical laws regarding refraction, Johannes Kepler (1571-1630) would mathematically express the actions of telescopes and microscopes, and Christian Huygens (1629-1630) the movement of light waves through air and denser mediums such as glass.

     One of the first recorded uses of a lens to magnify the image of a small object was by the Italian astronomer Galileo Galilei (1564-1642).  An Englishman traveling to Italy with Galileo in 1610 related:

          "I heard Galileo himself narrate how he distinguished perfectly with his optic lens the
           organs of motion and sense in the smaller animals.  Especially he observed in
           a certain insect that each eye is covered by a thick membrane, perforated with
           holes like the iron visor of a warrior, thus affording passage to the images of visible
           things."*

     While Galileo is best known for his studies of astronomy and the use of the telescope to make images far away appear much closer, it is obvious that he also understood that the same types of lenses could make small objects appear larger.  This idea was central to the development of early microscopes.

     Probably the best known of the early microscopists was Antony van Leewenhoek (1632-1723).  van Leewenhoek, a Dutch government official of minor status, was fascinated with lenses and spent much of his spare time making microscopes.  He was well aware of the limitations of early multiple lens microscopes which suffered from aberrations due to poor construction, and was unique among his contemporaries in that his microscopes used single lenses.  He found that small, single lenses could be finely ground and provided him with much sharper focus of the images he viewed.  van Leewenhoek jealously guarded his microscopes, never selling them and only rarely giving them away.  Over the course of his life, he used these devices to examine tissues, blood, sperm, insects, protozoa (which he referred to as "animalcules"), and in 1683, bacteria.  His drawings of common bacterial shapes, rods and cocci, are very good approximations of actual forms known today.  In fact, the impact van Leewenhoek had on the study of microorganisms, has led many to consider him the father of modern microscopy.
 

 The Cell Theory

     Another classical microscopist whose work was to have a major impact on the science of microbiology was Robert Hooke (1632-1723).  In 1665, Hooke published a major monograph entitled Micrographia, wherein he described various observations of objects made with a microscope composed of a multiple lens system.  The accuracy of the figures drawn by Hooke were viewed as a standard for many generations.  One of the illustrations is of a piece of cork, showing its construction as that of multiple, hollow chambers which he described as cellulae, which is Latin for "small rooms".  This term would later be shortened to cell by late eighteenth-century microbiologists and used to describe the most basic complete part of all living things.

     By the mid-nineteenth century, the concept of the cell was becoming well established as biological doctrine.  In 1838 Matthias Schleiden (1804-1881) proposed that all plants are composed of cells:

           "Plants, developed in any higher degree, are aggregates of fully individualized,
            independent, separate beings, namely the cells themselves... Each cell leads a double life;
            one independent, pertaining to its own development alone; the other incidental, as an
            integral part of a plant.  The vital process of the individual cells, however, form the
            first indispensable and fundamental basis for both vegetable physiology and
            comparative physiology in general.  Thus the primary question is, what is the origin
            of this peculiar little organism, the cell?"

                                             Abridged excerpt from "On Phytogenesis" in
                                            Archiv für Anatomie und Physiologie
                                             Johannes Müller    1838*
 
 

     One year later, Theodor Schwann (1810-1882) would extend this concept to animals while describing cartilages:

          "These tissues originate from cells, which correspond in every respect to those
           of vegetables.  During development, also, these cells manifest phenomena
           analogous to those of plants.  The great barrier between animal and vegetable
           kingdoms, vis. diversity of ultimate structure, thus vanishes."
                                            Abridged excerpt from Microscopical
                                           Researches on the Similarity in Structure
                                           and Growth of Animals and Plants
                                            Theodor Schwann  1839*
 

     With the concept of the cell firmly established in regards to microbes, plants, and animals, the next major step was to clarify how cells actually arise.  This question was answered by the German pathologist Rudolf Virchow (1821-1902), who, while studying the origin of disease in cells and tissues, forwarded the idea of self-replication.  Virchow wrote in his 1858 monograph Cell Pathologie:

        "Where a cell arises, there a cell must have been before, even as an animal can
         come from an animal, a plant from nothing but a plant.  Thus in the whole series
         of living things there rules an eternal law of continuous development.  There is
         no discontinuity nor can any developed tissue be traced back to anything but a cell."*

      This line of reasoning would lead Virchow to coin the aphorism, Omnis cellula e cellula, which literally translated means "Every cell from a cell".  In time, the combined works of Hooke, Schleiden, Schwann, and Virchow, could be condensed into a single statement of belief called the cell theory which says:  (1) all living things are composed of cells, and (2) all cells arise from other cells.
This theory is universally accepted today.
 

The Germ Theory of Disease

     The idea that disease could be caused by organisms which are too small to be seen was not a new one, even at the time the cell theory was becoming firmly established as doctrine.  Seventeen-century microscopists were well aware of certain organisms which could cause some illnesses, and even early Egyptians learned to treat some diseases with moldy bread, though it is unlikely that they truely understood the nature of what they were treating.  Agostino Bassi (1773- 1856) demonstrated in 1835 that a disease of silkworms could be transmitted from moth to larvae, and also that the disease was caused by a fungus.  Jacob Henley (1809-1885) developed his theory in 1840 that many diseases could be caused by microorganisms, but much of his work was dismissed as scientific heresy.  In 1862, however, Louis Pasteur discovered that there were two microbial pathogens involved in the silkworm disease which was sweeping through the French silk industry, and formulated specific treatment regimes which corrected the problem.  Since Pasteur was somewhat of a scientific celebrity for his time, his theories carried much weight and were more widely accepted than those of Henley.  Meanwhile, the French microscopist Casimir Davaine (1812-1882) had proposed that small bodies he called "bacteridia" were always present in the bodies of sick animals, and Ferdinand Cohn (1828-1898) began to develop classification schemes for bacteria based on the effect that each species had upon the various media in which they were grown.  Cohn's meticulous work was ultimately responsible for wide acceptance of bacteria as organisms, as is now considered to be the father of modern bacteriology.

      In Hungary, Ignaz Phillip Semmelweis (1818- 1865) became aware of the fact that many otherwise healthy women were developing fatal septic puerperal or "childbirth" fever when they gave birth in the hospital he attended.  Hospitals of the day were generally very dirty places, and it was not uncommon for doctors to move from one patient to another, or from the autopsy rooms directly to the maternity rooms without ever removing their stained overcoats or washing the blood and bodily secretions from their hands.  Semmelweis instituted hand washing and the trimming of beards and mustaches among his attendants, and consequently the incidence of mortality due to puerperal fever declined significantly.  Unfortunately, he was scorned for this departure from the conventional norms of the day and his policies were generally ignored by most physicians.

  One individual who did realize the efficacy of a cleanliness regime such as that proposed by Semmelweis was Joseph Lister (1827-1912), an English surgeon.  Lister was well aware of the works of Pasteur and Tyndall, so he understood that bacteria could be transmitted very easily by air, fluid, or hand.  To counter the spread of infectious agents, he began the process of treating the hands of doctors before surgery and spraying into the air during surgery a 5% solution of phenol (carbolic acid).  This reduced post-surgical mortality dramatically and allowed doctors to perform complex procedures they were previously hesitant to attempt.  Lister's antiseptic technique was a breakthrough in medicine and would ultimately lead to the development of the modern aseptic surgical practices used today.

     Robert Koch (1843-1910), a student of Henle, was a country doctor who in 1872 was faced with the problem of an outbreak of the disease anthrax in many of the cattle in his district.  Koch lacked the resources of his contemporaries at universities, so he began to develop his own specific techniques to determine the  etiologic (causative) agent of the disease.  By studying various tissues harvested from infected cattle, he discovered the presence of a bacterium, Bacillus anthracis, in all of the cases.  He also found that if he infected mice with the microbe, they too would develop symptoms of the disease.  Through microscopic examination, he realized that within many of the bacteria and at the ends of long chains of bacteria there were small structures, spores, which were resistant to heat, drying, and treatment by chemical agents.  In 1876, Koch published The causative factors of anthrax based on the development of Bacillus Antracis, which outlined his findings and for the first time clearly established the relationship between bacteria and disease.  With the broad acceptance of this publication, Koch found himself able to devote his time fully to the study of microbiology.  He soon developed staining techniques to more readily visualize bacterial cells, as well as cultivation techniques utilizing agar-based media, which allowed him to cultivate axenic (pure) cultures of specific bacteria for study.  Moreover, Koch developed a series of practical steps used for the identification of the etiologic agents of disease, which are known today as Koch's Postulates:

 1. The suspected pathogen must be present in all individuals suffering from the disease, but
     absent from all healthy individuals.

 2. The microorganism must be grown outside of the body of the diseased individual, in
     pure culture free of any other species of organism.

 3. When the pure culture of the suspected pathogen is injected into the body of a healthy
     laboratory animal, the animal should exhibit the symptoms of the disease.

 4. The microorganism must be re-isolated from the body of the laboratory animal and
     shown to be identical to the original suspected pathogen.

     Following these methods, Koch was able to establish the etiologic agents of tuberculosis in 1882, and cholera in 1883.  Pasteur would later repeat the techniques used by Koch to isolate the agent of cholera in chickens, and then use this agent to produce immunity in his test subjects.
 

Immunity from Disease

     Since early recorded history, it has been generally understood that if an individual developed a particular illness, and if he recovered from that illness, he might never suffer from the same malady again.  The specific reasons behind this were not well understood.  The earliest attempt at utilizing this phenomenon was made by the English physician Edward Jenner (1749-1823).

     One of the major communicable diseases ravaging England during the eighteenth-century was smallpox.  It had previously been shown in the early 1720's by Lady Mary Whortley Montague that deliberate infection of a healthy individual with a mild strain of smallpox could lead to some degree of immunity to more virulent strains of the disease.  This meant, however, that exposure to a possibly fatal malady was necessary.  Jenner recognized that milk maids were very seldom infected with smallpox, but did occasionally develop cowpox, a mild disease which could cross from animal to human.  He reasoned that somehow, exposure to this less virulent disease conferred upon the milk maids immunity to smallpox, and decided to put this to the test.  In 1798, Jenner inoculated  9-year old Jimmy Pruitt with fluid from a cowpox pustule on the hand of a milkmaid, then after a sufficient time deliberately inoculated him again, this time with fluid from a smallpox pustule on a human.  The boy showed none of the symptoms of smallpox.  Jenner was subsequently awarded the first true medical grant to study this phenomenon, which he named vaccination, a term derived from the Latin vacca which means "cow".  Louis Pasteur would later use vaccination to treat fowl cholera and anthrax, both diseases caused by bacteria.  He found that if he injected old cultures of these organisms into test subjects, then injected the same subjects with fresh, virulent strains, the individuals showed resistance to such infection.  This concept, called attenuating, or stripping away virulent factors from potential pathogens without killing them, was to catch on and lead to the establishment of many new forms of vaccines.

     The exact nature of immunity from disease, however, was still unknown to science.  In the nineteenth century, two rival theories arose concerning this phenomenon, one proposed by the researcher Elias Metschnikoff (1845-1916), and another by Emil von Behring (1854-1917).  Metschnikoff proposed that potential pathogens entering the body were engulfed and destroyed by special cells called phagocytes, named from the Greek for "devouring cells".  Behring, however, proposed that immunity to disease is a purely humoral or fluid-based phenomenon, after finding that he could confer resistance to disease in one animal by injecting into its body serum from another animal.  Behring coined the term "antitoxin" to describe that property within serum which conferred such resistance.  Today, we know that resistance to disease arises as a consequence of both humoral and cell-based aspects of immunity, with both aspects working together and complementing one another.
 

Antimicrobial Therapy

 For many years, it had been known that some forms of chemical agents could be used to treat disease in humans.  The Sixteenth-Century physician Aureolus Paracelsus used metallic elements such as antimony for general infections and mercury to treat syphilis, and in the early Seventeenth-Century Thomas Sydenham utilized the bark of the cinchona tree to develop a treatment for malaria.  While these early treatments did have some therapeutic effect, the exact mechanism of their effectiveness was unknown, and many were as toxic to the infected individual as they were to the pathogens they were used to inhibit or kill.

     The possibility of treating infection with chemical agents was vigorously pursued by the German researcher Paul Erlich (1854-1915).  After learning of the effects of staining on various bacteria by his cousin, Carl Weigert, who pioneered the process of differential staining of bacteria, Erlich developed the concept of selective toxicity, which means that some chemical agents have the ability to inhibit or kill microorganisms without causing substantial damage to their host.  Erlich would use this basic concept to develop a vaccine against diphtheria in 1904, and later to treat syphilis.  Erlich was a meticulous researcher who examined the effects of many different chemical agents on bacteria while searching for what he called a "magic bullet" for each.  In the case of syphilis, he and his research team tried 605 different agents before discovering one he called salvarsan 606, an arsenic derivative which was successful.  Erlich's work established a new field, chemotherapy, which is the basis for many forms of antimicrobial agents today.

     Along similar lines, the German physician Gerhard Domagk (1895-1964) discovered that the orange dye protonsil was an effective treatment for staphylococcal infections.  It would later be found that the active ingredients in this dye were sulfanilamide compounds, later to be referred to as "sulfa drugs", as well as the antimicrobial agent isoniazid.  Interestingly, Domagk would be awarded the Nobel Prize for medicine for his discovery, but Adolf Hitler prevented him from receiving the credit he deserved.

     Alexander Fleming (1881-1955), a British bacteriologist, was the first individual to recognize that some organisms exhibit antibiosis, or the ability to produce natural compounds which inhibit the growth of competitors.  In 1928, while researching influenza, Fleming found a mold growing on a culture plate containing the bacterium Staphylococcus aureus.  He recognized that the bacterium was inhibited in its growth by the mold, since there was a zone of clearing where no bacteria grew near the fungal mycelium.  He described the mold, Penicillium notatum, and the phenomenon due to its growth, then put it aside.  Later, the researchers Howard Florey and Ernst Chain would develop techniques for the cultivation of Penicillium mold and the purification of the first widely available antibiotic, penicillin G.
 

Modern Microbiology

 Since the 1940s, knowledge of microbiology has expanded at an ever increasing rate due to advances in microscopy, biochemistry, and genetics research.  In 1953, James D. Watson and Francis H.C.Crick defined the structure of the DNA molecule.  In 1956, F. Jacob and E.L. Wollman discovered the circular structure of the bacterial chromosome.  Two years later, M. Meselson and F. W. Stahl described the semi-conservative nature of DNA replication.  In 1960, F. Jacob and J. Monod proposed the operon theory of protein synthesis in bacteria.  Based on the work of these and many other researchers, scientists are now able not only to identify bacteria based on their metabolic characteristics, but are also capable of sequencing the entire genetic makeup of individual species.  Greater understanding of the disease process has enabled researchers to develop hundreds of different chemotheraputic agents which target individual or whole classes of microbes, and the use of aseptic technique now allows doctors to perform operations which would have been impossible less than one hundred years ago.  However, it is becoming obvious that new threats to the health of humanity are arising every day, both from the improper and indiscriminate use of antibiotics, and from new, unknown bacterial and viral diseases which have appeared in the last twenty years.  Viral diseases such as the HIV, Ebola, and Hantaviruses continue to puzzle scientists in terms of their origins and pathologies, and many diseases such as smallpox, cholera, and diptheria which were popularly believed to be extinct have now re-emerged.  The future will require a new breed of microbiologist who has both a grasp of the past and present and is willing to explore currently nonconventional avenues to find strategies for the prevention and treatment of disease.

*All quotes have been taken from Charles Singer's A History of Biology:  A General Introduction to the Study of Living Things.
  1951 New York: Henry Schuman, Pub.
 
 

Critical Thinking Questions

1.  Louis Pasteur finally ended the debate on abiogenesis with his classic "bent flask"
     experiment. Suppose that you were faced with the same problem, but knew
     nothing about Pasteur's work.  Create an experiment you could perform which
     would disprove the idea of spontaneous generation.
 
 
 
 
 
 

2.  Consider the postulates developed by Robert Koch for the identification of
     the etiologic agent of infectious disease.  Can you think of any exceptions to
     these postulates (i.e. examples of microorganisms which would violate Koch's
     methods)?
 
 
 

Test Yourself - This is a self-scoring quiz.
 

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