Which Scientist Discovered Heat Resistant Bacterial Spores

Abstract

1 Introduction

Progress in science has never been a continuous process. Discoveries, revolutionary ideas, and new concepts were very often neglected, misunderstood, or attacked if they did not follow the mainstream of the time. Although bacteria were discovered by observation under the microscope in the 17th century, they were believed to generate spontaneously and to be transformed into other morphological and physiological types (pleomorphism). It was not clear whether bacteria and other infusoria were the cause or the products of biological or chemical processes. Discussions on controversial concepts initiated experimental work to prove or disprove theories. Rational empiricism slowly overcame speculative reasoning.

2 The roots of modern science originated in the 17th and 18th centuries

In the 17th century, people began to trust the testimony of nature more than the testimony of human ideas. A scientist proceeds from the evidence of his own observation, but also from the basis of human written authority. Modern empiricism was borne on the concept that proper knowledge ought to be derived from a direct sensory experience of reality in nature. The inductive method for scientific work in which the conclusion is based on observations and measurements was proposed by Francis Bacon (1561-1626). The classical method of deduction, which solved scientific problems by sharp reasoning, was still in use then, but empirical research was favored. René Descartes (1596-1650) propagated that conclusions were only valid if they could be mathematically proven (in Principles of Philosophy, published in 1644). It was still a long road to the modern ideas that there is no absolute truth in biology and that a working hypothesis disproved by experimental evidence has to be replaced by a new hypothesis.

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Galileo Galilei’s Discourse Concerning Two New Sciences (1620), Robert Boyle’s Excellence and Grounds of the Mechanical Hypothesis (1666), and Isaac Newton’s (1643-1727) Mathematical Principles of Natural Philosophy (1687) introduced a mechanistic view of nature. Physical forces were put forth as the cause of biological processes. There was no dissonance between reductive causal explanation, the omnipresence of God and his part in the vital principle, and mathematical abstraction. In addition to the material world, the spiritual world was stressed by Baruch Spinoza (1632-1677).

Gottfried Wilhelm Leibniz (1646-1716), who reacted positively to the discovery of the world of microorganisms, tried to harmonize religious belief with the aspects of the new science. The animalcules were designated as a very low order of monads. The philosophers of that time found in the revelations of the microscope a way to combine the natural and supernatural worlds and to place the knowledge of nature within theological space [1]. The mechanistic-materialistic view was supported and extended by Georges Louis Leclerc Comte de Buffon (1707-1788), who published a universal history of nature with descriptions from minerals up to humans (Histoire Naturelle, Général et Particulière, 1749-1804, 54 volumes, completed by Etienne de Lacépède after Buffon’s death). Buffon stressed the importance of fossils as witnesses of earlier periods of life on earth, but he denied the descent of extant organisms from those of earlier periods because bastards are sterile and evidence of intermediates between present and extinct forms was lacking. He believed the Newtonian concept that organic molecules from the decomposition of organisms generate or can be incorporated into a new organism [2]. In his work both the inductive method and the deductive method were used. By the end of the 17th century, even those naturalists and philosophers influenced by the mechanical perception of nature doubted Descartes’ effort to derive the generation of animals from a mechanistic nature [3].

Scientists who preferred the inductive method were confronted with problems such as how precarious instrumentally mediated experience could be and how much work was required to declare observations as reliable. Another problem accompanying the more modern use of individual sensory experience was the evaluation of traditionally established knowledge [1, 3-5].

Although the inductive method was used more and more in research, philosophers and many scientists, influenced by philosophical ideas, believed until far into the 19th century that they could solve biological problems by reasoning and explain physiological functions by unclearly defined abstract terms like ‘vital forces’. Immanuel Kant (1724-1804) believed that in our thoughts we pass from a mechanistic view of the parts to a teleological view of the whole, and we cannot separate these classes of view; there is a hidden basic principle of nature which unites the mechanistic and teleological views. Georg Christoph Lichtenberg (1742-1799), a mathematician and physicist in Göttingen, should be mentioned because he personified the experimenter who was critical and skeptical of his own results and rejected any speculative philosophy. He represented an example of the English empiricism in the age of enlightenment.

3 The progress of biology in the 19th century

In the 19th century, many biologists made an imperfect fusion of the Kantian scheme with materialism, as for example by the ‘Naturphilosophen’ such as Lorenz Oken (1779-1851), who contributed considerably to embryology, but who tried to construct a biology that could reflect the action of the human mind in the animal kingdom and who considered ‘natural sciences as the science of the eternal transubstantiation of God in the world’ (1848); Gottfried D. Nees von Esenbeck (1776-1858); and Carl Gustav Carus (1789-1869), who made careful comparative studies in different fields, but tried to explain biological phenomena by teleological principles. These men proposed ‘ideal forms’ and linked them with the conception of the purpose that is inherent in living things. The ideas of Naturphilosophie, which dominated in the first half of the 19th century, especially in Germany, were replaced by the unifying idea of natural sciences in order to discover the laws of nature and to rule humanity. One exponent of this thinking was the German pathologist and anthropologist Rudolf Virchow (1821-1902) [5, 6].

3.1 The progress in chemistry

In addition to the mechanistic view of biological processes, the beginning of chemical analysis was important for the progress in biological research. Antoine Laurent Lavoisier (1743-1794) disproved the phlogiston theory of Georg Ernst Stahl (1660-1734) by determining the weights of products from chemical reactions. Oxygen, dinitrogen, and carbon dioxide were discovered as components of air. Joseph Gay-Lussac (1778-1850) isolated the metals of the alkaline-earth group and formulated the law of combining volumes of gases. In 1811, Amadeo Avogadro described the distinction between molecules and atoms and proposed the concept that equal volumes of different gases contain the same number of particles under the same physical conditions. The modern system of chemical symbols and formulas was developed and many new elements were discovered by Jöns Berzelius (1779-1848). He also coined the term ‘catalysis’ for a process in which a compound affects the velocity of a chemical reaction, but remains unchanged and does not contribute to the substrate or products of the reaction. Friedrich Wöhler (1800-1882) and Justus von Liebig (1803-1873) strongly influenced biology through their analysis and synthesis of organic compounds and by disproving the role of a vital power in the synthesis of organic compounds. They refuted, however, the role of microorganisms in fermentation and putrefaction and proposed, instead of ‘the metamorphosis’ by an organic product of decomposition, the ‘ferment’.

Although bioenergetics and thermodynamics were not included in textbooks until late in the 19th century and thermodynamic calculations were not considered for metabolic processes, the undefined and obscure term ‘vital force’ was replaced by the understanding that activities of living things require the performance of work, whether chemical, mechanical, osmotic, or electric [7]. The theory of heat and conservation and the correlation of energy was proposed by Julius Robert Mayer in 1845 and the law of the conservation of energy and the calculation of heat units were proposed by Hermann L. Helmholtz in 1846 and James Prescott Joule in 1843. Scientists slowly became aware that energy is supplied by metabolism, respiration, fermentation, and photosynthesis, but the problem of energy coupling, i.e. the linkage between energy-yielding and energy-consuming processes, was not resolved before the 1940s and 1960s.

3.2 Anatomy, microscopy, developmental cell biology, and sexuality

In the 16th to 18th centuries, the great diversity of plants and animals was recognized and the anatomy of humans, animals, and plants was described in numerous comprehensive articles.

The first microscopes consisting of one or two optical lenses were build by Johannes and Zacharias Janssen about 1590. The propagation of light and its reflection and refraction by an optical lens has been known since the work of Christian Huygens (1629-1695). He constructed simple microscopes and telescopes. The magnification of these microscopes was low, and the pictures of objects were affected by the chromatic and spherical aberration of their lenses because the light rays of various wavelengths do not focus in the same plane. It is remarkable that Marcello Malpighi (1628-1694), Nehemiah Grew (1628-1711), Antonie van Leeuwenhoek (1632-1723), and Robert Hooke (1635-1703) observed and described bacteria, protozoa, fungi, spermatozoa, erythrocytes, and tissues of plants and animals in fine detail using these primitive instruments. Leonhard Euler (1707-1783) postulated and several scientists such as G.B. Amici and A. Chevalier constructed the first achromatic lenses by combining lenses of different refraction indices. The objectives and oculars of the commercially produced microscopes were, until late in the 19th century, of variable quality because they were produced by empirical methods of trial and error. A progressive step was taken when the physicist Ernst Abbe (1840-1905) developed a theory of the image formation in the light microscope and constructed, in cooperation with the mechanic Carl Zeiss (1816-1905) and the producer of optical glass Friedrich Otto Schott (1851-1935), oil-immersion lenses with a high numerical aperture and the Abbe condenser for optimal illumination of the specimen field. The problem of spherical aberration, which caused blurred figures in the periphery of the microscope field of view, was solved by a combination of different lenses. These new improved microscopes were made popular in the cell biology and microbiology institutes about 1877-1878 by Ferdinand Cohn and Robert Koch, who tested these objectives and Abbe’s condenser [8, 9]. Since then, microscopes fitted with oil-immersion lenses that bring about a maximal resolution of 0.2 μm have been available. The documentation of bacteria was decisively improved by Robert Koch (1843-1910). He developed the method of staining smears in cooperation with C. Weigert [10] and the method of fixation of bacteria on cover slips, and introduced microphotography [11, 12] using a heliostat for illumination [11-14]. The difficult problems in the preparation of microphotographs were vividly described by Heymann [13].

The cells as building stones of tissues and organisms were described by C.F. Wolff. The great period of cell biology began in the 1840s with the availability of an improved microscope, the knowledge of comparative histology increased, and new thoughts on the role of cells grew [15]. Johannes E. Purkinje (1787-1869) was the first to use the term protoplasm [16] and proposed the idea of the similarity of animal and plant cells. Hugo von Mohl (1805-1872) and Matthias J. Schleiden (1804-1881) were recognized as the founders of the cell theory; they considered the cell as an independent living entity of all organisms [17-19]. Initially the protoplasm was considered as a very simple mucous material. In the 1880s, the nucleus was shown to be an indispensable constituent of animal and plant cells, and mitotic cell division was described by E. Strasburger, Francis Balfour, L. Auerbach, and W. Flemming.

Microorganisms were believed to be very variable (pleomorphic) and to generate spontaneously. In the period of flourishing cell biology around 1840, many scientists began to study the developmental history of lower algae, fungi, and protozoa, and later of bacteria. Ferdinand Cohn (1828-1898) investigated the unicellular algae Protococcus pluvialis Kützing and Stephanosphaera pluvialis and elucidated the different stages of development and the difference between vegetative and generative multiplication of swarm cells (macrogonidia and isogametes) [20-24]. About the same time, but independently, the sexuality of algae was discovered in 1854 by Gustave Thuret in Fucus, of Vaucheria in 1855 by Nathanael Pringsheim (1824-1894), and of Sphaeroplea annulina and Oedogonium in 1855/1856 by Cohn [22, 24]. The fecundation of the egg cell in the oogonium by the spermatozoids or the fusion of isogametes in cryptogamae, algae, and fungi was carefully studied by Cohn, Heinrich Anton de Bary, Thuret, Pringsheim, and others [19]. It was concluded that sexuality is a peculiarity not only of higher but also of lower organisms. The discovery by Cohn [25, 26] of the complex developmental cycle, functional differentiation, and sexual reproduction of Volvox globator and many other microorganisms was not only interesting from the point of cell biology, but also important for modern taxonomy and physiology. The concept that shape and function of each higher organism is based on a special plan was developed by the comparative anatomy studies in the 17th and 18th centuries. The progress in chemistry and comparative cell biology extended this concept to cells as the building stones of all organisms which are structurally and functionally differentiated during the development of the organism.

3.3 The development of the evolutionary view in biology

Several scientists of the 17th and 18th centuries became aware that extant life forms were organized differently than those of earlier periods of the Earth’s history. It was also realized that most organisms live in restricted areas, in natural habitats. The question of the origin of species was realized and the descent of species from common ancestors was discussed, but the static view of nature and the belief that all organisms could be traced back to creation or different forms of spontaneous generation, such as abiogenesis or heterogenesis, dominated [3, 27, 28]. Species were believed to be invariable.

Jean Baptiste Antoine de Monet Chevalier de Lamarck (1744-1828) was one of the first to explain the multiplicity of forms of organization and their gradation from primitive to highly developed species by a process of evolution [28, 29]. Impressed by the comprehensive comparative studies of fossils and living organisms, he postulated that the fossil species are ancestors of the living species. Lamarck proposed that the environmental conditions changed over long periods and that low to high complexity evolved by an inherent potential and by adaptation to the changed environmental conditions. He believed that the acquired properties were transmitted to the next generation. Although he did not explain the mechanism, his theory of evolutionary change replaced the static view of nature.

Georges Léopold Cuvier (1769-1832) contributed to the theory of evolution with comprehensive comparative studies on the anatomy of vertebrates and invertebrates and with paleontological studies, but continued to believe the constancy of species. He and the geologist Charles Lyell believed that extinction of species was caused by changes in environmental conditions during geological periods and that new species originated discontinuously by creation, spontaneous generation, or sudden changes.

Charles Robert Darwin (1809-1882) founded his theory of the evolution on the basis of his own studies and the numerous published observations of comparative anatomy of living and fossil organisms [30]. He concluded that all organisms have a common origin. Darwin assumed a continuous formation of a large and inexhaustible supply of genetic, i.e. inheritable, variations. In subpopulations of species, living in a separate habitat, natural selection caused diversity even within a species. Individuals and populations formed that changed their features slowly from that of the original species. New species originate from varieties. Natural selection was, in the view of Darwin and Alfred Russel Wallace (1823-1913), not an accidental process, but was caused by differential success in reproduction and competition within the population of the organisms and was determined by the interactions with the specific physical, chemical, and biological conditions of its habitat. New varieties optimally adapted to their surroundings survived and dominated in their habitat; less adapted varieties disappeared [28, 30, 31]. The principles of natural selection, evolution, and origin of species were not accepted immediately by the scientific community of the 19th century, and the concept of spontaneous generation by abiogenesis was revived to explain the formation of the first organisms on Earth [6, 27].

4 The discovery of microorganisms and their first classification

4.1 The bacteria

The supposition that various diseases are caused by microorganisms was expressed several times in the early literature [29]. In 36 BC, Marcus Terentius Varro wrote that animals (animalia quaedam minuta) that cannot be followed by the eye were transferred through the air to other persons and caused serious diseases. It is clear from his publication that he described malaria, which is caused by the sporozoon Plasmodium and is spread by the mosquito Anopheles[32]. Girolamo Fracastoro (1478-1553) studied the ‘French disease’ syphilis and wrote in 1546 that the ‘contagion is an infection that passes from one thing to another’ by direct contact between two persons, by contaminated material, or over long distances. He also described the pathology of a contagious disease, presumably spotted fever caused by Rickettsiae [29, 33]. Athanasius Kircher (1602-1680) studied infectious diseases caused by a contagium animatum. In contrast to the medical doctors of his time, who believed that diseases are caused by putrefaction of the body humor or miasma, he observed ‘vermes’ in the blood or lymph nodes of people suffering from bubonic plague caused by Yersinia pestis. Presumably he did not see the bacteria, but rather particles in the tissues.

The first clear evidence for the existence of bacteria was given by Leeuwenhoek. He was an optician by hobby and constructed, as did many contemporaries, numerous microscopes. The important step forward came from his very careful and imaginative style of observation. In more than 200 letters to the Royal Society in London, which were published in the Transactions of the Royal Society, and in letters to Robert Hooke, he described different forms of bacteria, yeasts, and protozoa [1, 4, 34-36]. He also performed some simple experiments, e.g. he studied the influence of acetic acid on the mobility of bacteria, which he called animalcules, beesjes, or cleijne schepsels. The detailed description of bacteria from tooth plaque, water samples, and hay infusion is remarkable considering the low magnification and resolution of his simple instruments. The bacteria he saw were documented by drawings. The size of the bacteria was determined by comparison with grains of sand or erythrocytes. The movement of bacteria was described in detail. The new knowledge on the animalcules or vermes quickly circulated and initiated many microscopy studies in order to find infusoria in organic material or tissues from sick people [36]. The development of the idea of the contagium animatum up to the causal analysis of infectious diseases will be dealt with in Section 7.3.

Carl von Linné classified the microscopic organisms in the genus ‘chaos’ (1773-1776). Otto Friedrich Müller (1730-1784) criticized the scientists of this epoch for contemplating the infusoria without any critical characterization and classification. In his book Animalcula infusoria fluviatilia et marina, he classified the infusoria by morphological and biological criteria, such as movement, habitat, and formation of aggregates. From the 18 genera he proposed, only several characteristic types, especially Flagellata and Ciliata, can be identified. Bacteria, but also Protozoa, appear under the taxa Monas and Vibrio. He described 10 species of Monas and 31 species of Vibrio. The description of the infusoria was documented by figures [37]. The knowledge of the larger forms of infusoria, the protozoa and unicellular algae, was improved in the following decades, but the studies on bacteria concentrated on the question of their origin (see Section 6).

Christian Gottfried Ehrenberg used an improved microscope, fitted with achromatic combinations of lenses, to study the ‘Infusionsthierchen als vollkommene Organismen’ (infusoria-animalcules as complete organisms) [38]. The largest part of this book was devoted to the protozoa. The most simple organisms he observed were classified as Monadina and Vibrionia. The tail-less, lip-less, eye-less, most simple monadina were subdivided into sphere monads and rod monads. The genera Monas, Bacterium, Vibrio, Spirillum, Spirochaeta, and Spirodiscus were described, but the species were less well characterized. Félix Dujardin subdivided the bacteria, combined in the family Vibrioniens, into the genera Bacterium, Vibrio, and Spirillum [39]. Four species of Bacterium – B. termo, B. catenula, B. punctum, and B. triloculare – were described. The species Vibrio lineola, V. rugula, V. serpens, and V. bacillus were distinguished from Spirillum undula, S. volutans, and S. plicatile by their shape and movement. The organisms described by both authors cannot easily be identified by present taxonomic characteristics. The work of Maximilian Perty [40] did not improve the taxonomy. He confused the characterization by mixing up developmental stages with species. He subdivided the so-called animal-plants or Phytozoidia, into Filigera, Sporozoidia, and Lampozoidia, and further subdivided the latter into Vibrionida, Spirillina, and Bacterina. Allied to the Spirillina, the species Spirochaeta plicatilis, Spirillum volutans, Spirillum undula, and Spirillum rufum were mentioned. The Bacterina were subdivided into Vibrio, Bacterium, Metallacter and Sporonema.

4.2 The fungi

The large fruiting bodies of basidiomycetes and ascomycetes, conspicuous to the unaided eye, may have been known to man since primitive times. Humans have used fungi, often unknowingly, for fermentation in wine, beer, and bread making. Fungi were also used as food, drugs, or poison.

In the 16th and 17th centuries, the shape, appearance, and usefulness for man of numerous fungi were described, for example, by Charles de L’Ecluse (Clusius; 1526-1609) [41]. Gaspard Bauhin (1560-1624) was the first to discern genera and species in his detailed and illustrated description of plants. In his book, 2700 species of plants and 100 species of fungi were described. The fungi were separated into esculentii, noxii, and perniciosi [42]. Joseph Pitton de Tournefort (1656-1708) presented a hierarchical ordered system with detailed descriptions of genera [43]. Fungi were separated into six groups: (1) centrally stalked with cap; (2) centrally stalked without cap; (3) laterally stalked; (4) globose without stalk, including myxomycetes; (5) subterranean forms; and (6) corraloid forms. Developmental stages, e.g. teleutospores of rust or mycelium of fungi, as described by Robert Hooke, were not included.

How fungi propagated was not known, and they were believed to originate from decaying substances. The revolutionary idea that all plants produce seeds was proposed by Porta in 1590. He described spores, which he called seeds, isolated from numerous fungi. M. Malpighi pictured in his book of the anatomy of plants [44] sporophores, sterigmata, and spores, and speculated whether spores were units of propagation. Pier Antonio Micheli (1679-1737), although he never received an academic degree, made great progress in the description of cryptogamae. He introduced the names of many generic names, such as Mucor, Aspergillus, and Polyporus, and his descriptions of species are so detailed that they can be identified today. With a primitive microscope, he observed seeds (spores) and sporophores in many groups of fungi, and he cultured certain molds on pieces of fruit. He followed the germination of spores and the growth and development of fungi up to the fruiting bodies, and concluded that each fungus formed its own seeds and is reproduced only by its own kind [45]. Carl von Linné (1707-1778) included the described fungi as a class of their own in his book on species plantarum [46], where he established the binomial system of nomenclature. He did not, however, contribute to a better understanding of fungi.

In the second part of the 18th century, many detailed descriptions of fungi were published and illustrated with excellent drawings [47]. Phenomena of infected plants had been known since the classical period. Phytopathology, based on empirical research, was initiated by Mathieu Tillet (1714-1791). He studied loose smuts and hard smut of wheat and showed that they are infectious diseases. He observed that seeds (spores) from smutted kernels produce smutty wheat [48]. In 1767, Giovanni Targioni-Tozzetti, a disciple of Micheli, described the infection of plants by germinating rust spores. He observed the penetration of the epidermis through the stomata by germ tubes and the development of the fungus inside the wheat plant [49]. In the same year, Felice Fontana published the results of his microscopy studies on the rust of grain. Bénédict Prévost (1755-1819), who was not familiar with these important observations, published in 1807 his detailed and careful studies on the germination of bunt or smut spores and the infection and development of the fungus in wheat plants. He observed that copper sulfate inhibits the germination of spores, and he demonstrated by field experiments that the disease can be controlled by soaking the wheat seeds in a solution of copper sulfate [50]. Although this method was not widely accepted at that time, it was a precursor of the Bordeaux mixture, introduced by Pierre Millardet in 1885, which became in combination with the lime-sulfur solution the world’s outstanding preventive fungicide, used at first to fight downy mildew on vine leaves caused by Plasmopara viticola[51].

A new period of research in mycology began with Heinrich Anton de Bary (1831-1888). He and many of his students and coworkers, such as O. Brefeld, E. Fischer, A. Meyer, P. Millardet, and M. Woronin, published important papers in the field of sexual and asexual reproduction, development, and parasitism of fungi [52-55, 60]. In 1893, Pierre Dangeard observed nuclear fusion in the teleutospores of a rust fungus, and in 1894 in Peziza. He interpreted this process correctly as a fecundation. This progress in developmental biology was the basis of a modern taxonomy of fungi. Ferdinand Cohn studied the development of the zygomycete Pilobolus crystallinus and of the entomophthoracea Empusa muscae [56, 57].

Johann Schönlein in 1839 and David Gruby in 1841 described Trichophyton and Candida albicans as infectious fungal parasites of man and founded the field of medical mycology [29].

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The first systematics of fungi was published by Christian Persoon [58]. He understood that mushrooms are only fruiting bodies and are not the whole plant. He recognized two types of fruiting bodies: those in which the hymenium is uncovered during maturation of the spores and those in which the hymenium is enclosed, e.g. the puffball (bovist). He established a herbarium containing the type species. The fungi known at that time were divided into 71 genera. Other systematic treatments of fungi were published by Elias Magnus Fries [59], and in 1837-1854 by August Corda [29]. These early systematic studies were based exclusively on morphological data. De Bary emphasized the importance of the developmental history and of the sexuality for classification of fungi [53].

Nutritional physiology and the development of exact methods to analyze growth and nutrition of fungi were founded by Jules Raulin, who elucidated the mineral requirements of Aspergillus niger[61]. The growth of fungi on a mineral medium completed with an organic carbon source was introduced by Louis Pasteur, but Raulin was the first to determine quantitatively the growth of fungi and the consumption of the nutrients. He recognized that besides the macroelements and one organic carbon source, trace elements were essential for the growth of Aspergillus, but their exact analysis required improved methods for purification, which were not available at that time.

4.3 The protozoa

The protozoa, called ‘infusoria’, have been described in many monographs since the 17th century. Christian Gottfried Ehrenberg (1795-1876) was well known for his detailed and comprehensive description of more than 500 species [38]. He observed that the small animals take up particles of carmin or indigo into vesicle-like structures, which he called stomach. He proposed the concept that protozoa have complex internal structures similar to those of higher organisms. Felix Dujardin (1801-1862), who stressed the importance of the work of Ehrenberg, rejected this hypothesis as most of his contemporaries did. Dujardin improved the systematics of protozoa and observed that sodium phosphate, ammonium oxalate, sodium bicarbonate, and ammonium nitrate were used by the infusoria as nutrients [39].

5 The concepts of taxonomy

The comparative anatomy studies in the 17th and 18th centuries brought together a comprehensive knowledge of the shape, structure, and organization of organisms. The great diversity in the world of living beings, the purely practical need to bring order into the richness of life, and the desire to investigate the perfect harmony of nature and its diversity were motives to study systematics. In the era of Linnaeus, systematics had enormous prestige and dominated all other contemporary research. Linnaeus and his contemporaries believed that genera and higher taxa are creations of God and that therefore his systematics represented a natural system. This systematics was based on essential properties and originated from creationist thinking in the absence of an evolutionary theory (essentialism). The principle of logical downward division is based on the similarity of organisms and flowed from the higher to the lower taxa using the method of dichotomy. It was a purely descriptive work, but it was a rich source of information. Linné not only introduced binary nomenclature, he also completed the species description by adding remarks on the habitats of the species.

The quite different systems which originated in the 17th and 18th centuries were influenced by the choice of the characteristics used for the first division [28]. Even within a system, the type of characteristic was changed, e.g. from fructification to vegetative growth, or from morphological to physiological features. It was discussed whether one should use only a single key characteristic or multiple characteristics; whether one could use characteristics other than morphological characteristics for classification, e.g. physiological and ecological features; and whether the characters should be weighted. The refined and extended knowledge of organisms living in different parts of the world and the revolution in philosophical thinking made the downward classification of essentialism unsuitable for classification. Practical considerations led to the adaptation of an upward classification of empirical grouping using numerous characteristics. This method started with the characterization of species, followed by sorting of species into groups of similar ones, and combining these groups into a hierarchy of higher taxa [28]. In 1772, Adanson introduced the use of multiple characteristics for classification. He recognized that different characteristics have different taxonomic significance.

Taxonomy was revolutionized in the 19th century by Charles Darwin, the founder of evolutionary taxonomy. Darwin explained why groups of species are related to each other. In the thirteenth chapter of The Origin of Species[30], he developed the theory of classification. His theory of common descent provided reasons for the degrees of similarity among organisms and an explanation for the hierarchy and for the homogeneity of taxa in a natural classification. Darwin also discussed methods and difficulties of classification. He stressed that true classification is genealogical and, therefore, the taxonomical value of all characters has to be weighted. Similarities due to descent have to be separated from similarities due to convergence. However, the Darwinian revolution had only a minor impact on the methodology of classification. Upward classification had already been introduced before Darwin [28].

The classification of microorganisms, especially of higher taxa, was improved in the 19th century by the discovery of sexuality and of the development of fungi, lower algae, and protozoa. In earlier times, very often different stages in the life cycle or zoospores were described as different species or interpreted as polymorphy. The discovery of the ascus and the basidium and their role as meiosporangia was decisive for the grouping of ascomycetes and basidiomycetes.

5.1 The species problem in bacteriology and new concepts for classification

Bacteria have been known since the early observations of Leeuwenhoek, and many studies were published after that time which describe ‘small animals’ or ‘infusoria’ as contagion (Jakob Henle (1809-1885), contagium vivum [62]) or as ‘ferment’ of butyric acid fermentation [63, 64]. Unfortunately, no scientist carefully isolated the particular microorganisms and studied them in their environment. The bacterial forms observed with the microscope were described as a new species without consideration of the forerunner. The work of Ehrenberg and Dujardin was an exception, but they did not characterize the species sufficiently. The problem of the origin of bacteria and of independent, distinct species was still not solved by 1850.

Ferdinand Cohn (1828-1898) stressed that in the field of bacteriological systematics, one has to start at point zero [65, 66]. His taxonomic studies were based on an excellent knowledge of the unicellular algae, lower fungi, protozoa, and bacteria. He noticed that the cellular organization and other structural details of bacteria could not be resolved, even when the bacteria were observed with the strongest oil-immersion objective of the microscope (the methods of phase-contrast microscopy and staining of bacteria were still not discovered) [65, 66]. Only a few characteristics were available for classification, and it was not known whether they are stable and species-specific stages of development, or variations caused by environmental conditions. Sexual reproduction of bacteria was unknown.

The isolation of single cell clones and the pure culture technique were slowly developed from the cultivation on solid media and enrichment cultures. The growth of colored bacteria on starch-containing food was described by Herrmann Hoffmann ([36], p. 110). Joseph Schroeter, a coworker of Cohn, transferred colonies of pigmented bacteria, grown on slices of cooked potato, to another piece of solid food to separate the colored from the colorless bacteria [67]. Clone cultures of fungi were obtained in the laboratories of de Bary and Brefeld by sowing single spores on solid media ([54], pp. 2 and 5; [60, 68]). Scientists became rapidly acquainted with the culture of bacteria on solid media and the technique was used in the laboratories of Cohn and Koch starting in about 1875 [36]. The separation of single cells by streaking bacteria on solidified gelatin was introduced by Koch in 1877 [12] and the use of plates with gelatin was introduced by Koch [12, 14, 69] and Esmarch [70, 71]. Frankland [72] and Petri [73] developed a small, practical culture chamber, the Petri dish, to keep the cultures free of contamination through the air. The introduction of agar as a solidifying agent greatly improved the isolation and culture of bacteria because agar is inert for most bacteria and is still solid at 37°C, the temperature at which most pathogenic bacteria were cultivated [74, 75].

Cohn noticed that species and genera of bacteria have meanings different than those of higher organisms, because generative propagation was not known. The classification of bacteria had to start from the characterization of ‘form-genera’ and ‘form-species’. The question whether these species are related to each other in their development, chemical features, and descent could only be answered in the future when new chemical methods became available [65, 66, 76]. From the beginning of his studies, Cohn was convinced that the kingdom of bacteria consisted of species with inherent characters. He defended this concept against Theodor Billroth (1829-1894) and many other contemporaries who believed that all spherical bacterial forms and all rod-shaped bacterial forms each belong to only one species of plants and have ‘only one form of life’ (‘eine einzige Lebensform’) which can adapt to different conditions of the environment and change their form accordingly (pleomorphy): Micro-, Meso-, Megacoccus and Micro-, Meso-, and Megabacteria. Billroth combined all genera proposed by Cohn in the polymorphic species Coccobacteria septica, except for Spirillum and Spirochaete, which he did not consider [77, 78]. Joseph Lister defended the idea that bacteria are generated from conidia of fungi and that they change their morphology during culture on different media [76, 79]. From the description of his experiments, it seems clear that he transferred a mixture of different organisms to new media and that specific microorganisms were selected during growth. During this time, several pathologists described microorganisms in different diseased tissues, but they did not isolate and characterize the bacteria [36]. Although they speculated that these organisms cause the illness, no experiments were undertaken to identify the organisms and to study their effect on the human body.

During a period of about 20 years Cohn and coworkers studied numerous distinctive characteristics of bacteria, such as cytological details, movement, growth under various conditions in mineral media substituted with one carbon source or on complex media, appearance of pigments, and the formation and germination of endospores (Fig. 1). The structure of flagella and the swarming phenomenon were detected, and the function of flagella was correctly interpreted [66, 67, 76, 80]. On the basis of these investigations and his being impressed by Darwin’s theory of origin, evolution, and selection of species, Cohn developed a new concept of bacterial classification. Bacteria were defined as mostly pigment-free cells of characteristic shape that multiply by cross division and live as single cells, filamentous cell chains, or cell aggregates. They contain a plasma membrane and sometimes refractile granules. They form a distinct kingdom of microorganisms that are discernible by heritable characteristics that allow the classification into distinct species with typical characteristics, which are transmitted to the following generation when bacteria multiply. Cohn proposed that within the species, varieties originate, which transmit their new features to the next generation. He was convinced that bacteria belong to the plant kingdom and that they are related to algae [66, 76, 80-82]. From his studies on development, he concluded that bacteria are closely related to the Phycochromaceae (Rabenhorst 1865), which were also known as Myxophyceae (Wallroth 1833), Schizophyceae, or Cyanophyceae (Sachs 1874); today they are named cyanobacteria ([83], p. 1711). Schizophyceae and Schizomyceae (bacteria) were combined in the group of Schizophyta (fission plants) [76, 80, 84, 85]. The close relationship between bacteria and Schizophyceae was exemplified by comparison of the chlorophyll- and phycocyan-containing Oscillatoria with the colorless Beggiatoa (Fig. 2). These two genera have the same shape and cellular organization and the same type of movement, which is a combination of gliding forwards and backwards, a rotation around the longitudinal axis, and a vivid bending of the trichomes. The Chroococcaceae were compared with Micrococcus and Bacterium, Merismopedia with Sarcina, and Spirulina with Spirillum. A relationship between groups of bacteria, phycochromaceen, florideen (red algae), and lichen was deduced mainly on the basis of the pigments chlorophyll, phycocyan, and phycoerythrin and the type of cell division, movement, and reproduction [84, 85]. Cohn supposed that the Phycochromaceae were early inhabitants of the earth because of their ability to grow in extreme habitats, their simple way of reproduction, and their fossil record. The fungi were considered as a group of microorganisms not related to the bacteria and phycochromaceen [76, 86]. Cohn classified the bacteria into: (I) Sphaerobacteria (sphere-shaped) with the genus Micrococcus, (II) Microbacteria (rod-like) with the genus Bacterium, (III) Desmobacteria (filamentous bacteria) with the genera Bacillus and Vibrio, and (IV) Spirobacteria (screw-like bacteria) with the genera Spirillum and Spirochaeta [76, 80, 81]. Micrococcus was divided into three groups of species based on the characteristics chromogen (pigmented), zymogen (fermenting), and contagion (pathogen). Bacterium termo was specified as the cause of putrefaction. Contagion and putrefaction were discerned as specific features. In the description of the screw-like bacteria, Cohn followed the names proposed by Ehrenberg: Spirochaeta plicatilis, Spirillum volutans, S. tenue, and S. undula. The bacteria were cultivated on synthetic media with various carbon sources or on complex media. The inheritance of features, e.g. pigmentation, was supported by their stability in following generations. The pigments were differentiated by solubility or insolubility in water and their color and were analyzed by simple chromatographic, spectroscopic, and chemical methods. The genus Sarcina [66, 76] was determined by the occurrence of division in three perpendicular planes.

The principles of Cohn’s bacterial taxonomy were not generally accepted. H. Karsten, A. Wiegand, Estor, Winternitz, and others still believed that bacteria originated from decaying plant and animal tissues ([36], p. 130) or spontaneously [27, 76]. E. Hallier supported the germ theory of infectious diseases, but he believed that the micrococci he isolated from pathogenic material were transformed into fungi [87]. The fungus theory of Hallier was strongly refuted by de Bary because of Hallier’s faulty experiments [88]. Hallier considered the succession of different organisms that he observed in his culture apparatus as stages of the same organism, which he regarded as genetically connected [87]. Billroth, Lister, Nägeli [89], and other contemporaries defended the opinion that the morphological and physiological differences between the species are caused by nutrition and other growth conditions [35, 36]. T.A. Edwin Klebs (1834-1913), a pathologist, agreed with the hypothesis that bacteria can be classified into different species, and he defended the germ theory of infectious diseases; in addition to micrococci and bacilli, he proposed microsporines and monadines [90, 91]. Ray Lankester observed enrichments of purple bacteria in decaying organic material and named the pigment bacteriopurpurin and the bacterium Bacterium rubescens[92]. He thought that all the bacteria that contained bacteriopurpurin but which had different shapes were phases of one and the same organism (pleomorphism) [93]. Cohn, Ehrenberg, Engelmann, Warming, N. Winogradsky, and Zopf described many species of purple bacteria (the name was coined by Engelmann) mainly on the basis of their morphology, pigmentation, physiology, and cell inclusions (Fig. 1) [38, 76, 80, 81].

An important achievement of Cohn was the species concept, founded on the hypothesis that distinct species-specific populations have several inheritable characteristics in common, which differentiate them from other species. The concept was solidified as soon as pure cultures of bacteria were isolated which had stable markers such as pathogenicity, e.g. Bacillus anthracis[94], or nitrification, e.g. Nitrosomonas [95-97]. The principle of upward classification based on invariable, multiple characteristics remained the method of choice for decades. The progress in methods to study bacteria and the increasing knowledge of the physiological and biochemical features of bacteria led not only to the identification of new species, but also to grouping of the species into higher taxa. The concept of bacteria as an independent group of microorganisms with different inheritable characteristics and their relationship to the Schizophyceae (Cyanobacteria) was confirmed and extended by R. Stanier and C.B. van Niel [98], who proposed the term prokaryotic cell organization, which differed from the organization of eukaryotic cells. This fruitful concept was quickly accepted by the scientific community and later extended by the discovery of archaebacteria as an independent group of prokaryotes distinct from eubacteria and cyanobacteria [99, 100].

The concept of a bacterial species, however, is still open to discussion. Since prokaryotes do not have the same form of sexuality as higher organisms, of which species are defined as groups of interbreeding natural populations that are reproductively isolated, a bacterial species has been regarded as “a collection of strains that share many features in common and differ considerably from other strains” [101]. This definition is unsatisfactory because it remains in the opinion of the taxonomist to define a new species. It is known that bacteria can exchange genetic material even over large phylogenetic distances, but the extent to which homologous recombination between host and foreign DNA takes place differs strongly. In modern bacterial taxonomy, morphological characteristics, which dominated the classification in the past century, are of minor importance. The weight of the genomic characterization has increased over that of the chemical composition of cell constituents, e.g. the components of the cell wall and metabolic products and pathways. It is recommended that strains which share at least approximately 70% nucleotide sequence identity and a difference of less than 5°C in the melting point of DNA/DNA duplexes belong to the same species [102]. This level of similarity leaves room for genomic and phenotypic differences due to the different life history of the strains, but simultaneously allows the combination of these strains in a species, which has for practical purposes enough common features. There are several reasons for not changing the present classification of prokaryotes on the basis of a rigorous use of this species definition: (i) not enough data on strain diversity and interspecies relationships are available, (ii) different criteria have been applied to identify species, (iii) very closely related strains are for diagnostic purposes still separate species, e.g. in the group of enteric bacteria, and (iv) evolutionary systematics, as proposed by C. Darwin, is the only way to come to a ‘true natural system’ of bacteria.

An increasingly robust map of evolutionary diversification has been compiled, and the progress in the construction of phylogenetic trees on the basis of true DNA/DNA homologies is impressive. The recognition of Archaea as a distinct kingdom of organisms has influenced thoughts on the evolutionary relationships among living organisms. This phylogenetic classification will be the final objective of systematics, although at present there is no general agreement about the domain concepts of C.R. Woese (Bacteria, Archaea, Eukarya), the five-kingdom classification of L. Margulis (Prokaryotes [Monera], Eukaryotes [Plants, Animals, Fungi, Protists]), or the division of prokaryotes in Monodermata, having a single ‘cell membrane’ (Gram-positive) and Didermata, having two ‘cell membranes’ (Gram-negative) [103]. The important role of prokaryotes in the evolution of life seems to be evident, but the origin of the different groups of eukaryotic and prokaryotic organisms and their evolutionary relationship remains open to further studies. The same is true for the species concept for bacteria. A phylogenetic classification is the final goal, but an artificial classification is still in use, even though we know that markers such as phototrophy, autotrophy, pathogenicity, and methanogenesis are not characteristic for a phylogenetic group [102, 103].

6 Spontaneous generation vs. evolution of microorganisms

The hypothesis of spontaneous generation of living beings from the elements or from putrefaction, decaying organic matter, and humidity was an old belief described by Aristotle and other philosophers and poets of antiquity that occupied the attention of many scientists over the centuries and adhered to a wide spectrum of philosophical views until modern times [1, 3, 4, 27, 29, 35, 104]. All proponents of spontaneous generation believed that living entities can arise suddenly by chance from inorganic matter (abiogenesis) or from organic matter, which was itself derived from organisms (heterogenesis), independently of any parents. Vitalists deny any possibility of abiogenesis by chance, while others, following the universality of strictly deterministic natural laws in the mechanistic philosophy of R. Descartes, believed in the abiogenetic way of spontaneous generation. The doctrine of spontaneous generation has long been considered as an inhibitor of scientific progress. It will be shown here by a few examples that the controversial discussion on this theory became finally a driving force of scientific progress because it initiated numerous experiments and resulted in new concepts and experiments [27].

The extensive studies on the anatomy of higher and lower plants and animals in the 17th century revealed that animals and plants developed from eggs and semen. Francesco Redi (1626-1697) discovered that putrefying matter is not the material from which animals generate, but a substrate on which animals deposit their eggs. He observed that flies lay their eggs on flesh and that maggots generate from eggs, and flies from maggots. Leeuwenhoek was convinced that no living things came from putrefaction, but that they derived from those created in the beginning [4]. He studied the development of insects and vermes from eggs. From his results the following questions arose: (1) is air needed to support life?, (2) do all animals come from eggs?, and (3) can seeds and eggs survive for indefinitely long periods? [1, 4].

Leeuwenhoek rejected the idea of spontaneous generation also for the microscopic organisms and believed that the animalcules were distributed by air in the form of seeds or germs, but he did not prove this hypothesis [4]. The distribution of germs by air was a controversial issue in the 19th century. Louis Joblot (1645-1723) also denied the doctrine of spontaneous generation, and this standing was considered to be contrary to all reason and religion. He experimented with heated infusions to see whether they could produce animalcules. He boiled fresh hay and distributed the infusion into two vessels, one of which was closed and the other was left open. After a considerable time he observed animalcules only in the open vessel [105]. Like Leeuwenhoek, he believed that the eggs of animalcules were carried by air into the uncovered vessel. John Turberville Needham (1713-1781) performed similar experiments, but he used closed and open vessels in which he heated meat extract. In both vessels a dense population of organisms was observed after several days of incubation. Needham repeatedly observed that after heating different kind of infusions and careful closing to avoid contamination from air, innumerable filaments swelled from an internal force and became perfect zoophytes or microscopic animalcules [106]. He and also Buffon concluded that in each living material is a vegetative force – a universal semen that can initiate new life in any organic substance. Buffon developed the idea that vitality is an indestructible property of living things [107].

Abbé Lazzaro Spallanzani (1729-1799) observed in infusions a sequence of different animalcules. In numerous experiments, he showed that heating prevents the appearance of animalcules in infusions if the flasks are sealed hermetically and the air in the bottle did not contain animalcules. He found that the heating period required to render an infusion sterile is variable, and he concluded that Needham’s infusions were contaminated by air [108]. Needham commented that in Spallanzani’s experiments, the prolonged heating destroyed the vegetative force and modified the air. In response to Needham’s objections, Spallanzani repeated his experiments and concluded that animalcules developed in flasks that were corked, but not in hermetically sealed flasks that were heated for 0.5-2 h. He also did experiments with flasks having capillary necks to avoid diminishing the ‘elasticity of the air’, as accused by Needham. Two types of animalcules were described: those of superior order, which were easily destroyed in 30 s at 100°C, and the other exceedingly minute organisms that sometimes survived boiling for 30 min. He also showed that boiling water is much more effective in sterilization than hot air and that boiling media for long periods did not prevent growth of animalcula [108, 109].

The opposition to Spallanzani and like-minded people rested on the adherence to the traditional 18th century mechanistic concept of spontaneous generation. Opponents of spontaneous generation were faced with the problem of the generalization of an experimental result: the statements that all organisms arise from parents or that no organism arises spontaneously from matter can be disproved for one organism, but neither can be proven with absolute certainty. Moreover, Spallanzani had several limitations; most importantly, his microscope was insufficient to see bacterium-sized objects. He attacked the doctrine of spontaneous generation from an a priori belief in the hypothesis that all living things arise from parents. Thus, the experiments of Spallanzani and the critical thoughts of Leeuwenhoek did not convince the adherents to the doctrine of spontaneous generation of animalcules, and the doctrine remained a widely held belief and was supported by many biologists, such as O.F. Müller, Treviranus, Lamarck, Kützing, and Dujardin far into the 19th century [27, 35, 36]. Although cell biologists and histologists provided more and more examples of organisms that arise from parents and argued by analogy that all living things were produced in the same way, the doctrine survived in a transformed view. Lamarck believed that spontaneous generation is necessary in order to understand the discontinuities in fossil records and the evolution from the lower forms on the escalator of life to the more complex higher organisms [104].

The spreading of microorganisms through the air was very often thought to be a source of contamination. F. Schulze [110] gassed heat-sterilized infusions with air sucked through concentrated sulfuric acid. No growth of organisms was observed until the vessel with the infusion was exposed to open air. Felix-Archimède Pouchet and Hughes Bennet, however, observed growth in the apparatus described by Schulze. J. Tyndall (1820-1893) noticed that the air has to pass slowly through the sulfuric acid; otherwise, the gas bubbles could transfer microorganisms. Theodor Schwann (1810-1882) designed an apparatus consisting of a flask that contained an infusion sterilized by boiling. Air was conducted through a heated glass tube before reaching the infusion. Such flasks kept for 6 weeks did not show any growth of microorganisms, but after opening the flask, the infusion became putrid. Schwann concluded that the germs or seeds of infusoria in the air were destroyed by heat. In the same apparatus, the flasks were filled with boiled sugar solution and yeast. In flasks with unheated air, the sugar was degraded, alcohol was formed, and the yeast cells grew ([35], pp. 86-87). A new principle of air sterilization was introduced by H. Schröder and T. von Dusch [111]. They filtered air through cotton-wool before passing it through the infusion. The filter trapped the germs, and no growth was observed in the heated infusion. Although these and other scientists showed experimentally that no animalcules developed in boiled infusions and therefore no spontaneous generation occurs if the air is free of germs, the number of opponents did not decrease.

One of the opponents was F.A. Pouchet (1800-1872), who began his experiments with the aim of proving spontaneous generation. He believed that life was generated by a vital force coming from pre-existing living matter (heterogenesis [27, 112]). According to his theory, the main factors of heterogenesis are organic matter, water, access of air, and a suitable temperature. He repeated the experiments of Schwann and Schulze, but obtained contrasting results. At this time, Louis Pasteur (1822-1895) extracted germs from the cotton-wool after filtration of air and observed them under the microscope, thereby demonstrating that germs are distributed through air [63]. The presence and distribution of microorganisms in the air was also studied in Cohn’s laboratory. In a simple set-up, air was sucked through a previously sterilized medium. The aerated medium was incubated at different temperatures, and the growing fungi and bacteria were studied microscopically [113]. Miflet showed that the number and type of microorganisms growing in the aerated media were dependent on the source of air (laboratory, garden, or open field). Similar experiments were performed by Pasteur and coworkers, who showed that air in the mountain region of the Alps at 2000 m altitude contained far fewer germs than air in locales in Paris [63, 104, 114]. Pasteur also concluded that alkaline infusions required a higher temperature or prolonged periods of boiling to destroy the germs than acidic infusions [104, 114, 115]. In order to avoid contamination, but to allow access of air to the infusion, he used flasks with a neck that had been heated and pulled out into a capillary and bent several times: the boiled infusion in these flasks remained sterile. The spontaneous-generation proponent Pouchet did not give up. He conducted an extensive series of experiments similar to Pasteur’s, but again came up with contrasting results. Finally, a commission of the Academy of Sciences, which was prejudiced, decided the discussion in favor of Pasteur. It was not seriously considered that Pasteur worked with yeast extract or other defined media, while Pouchet used hay infusions. Pasteur refuted the doctrine of spontaneous generation not only because of the results of his sterilization experiments, but also because of preconceived ideas that specific fermentations, such as butyric, alcoholic or lactic acid fermentation, are caused by specific microorganisms even when no oxygen is present and because of his anti-materialistic belief in a Creator God [63, 64, 104, 114-117].

The heterogenists and others continued to carry out different types of experiments to prove or disprove spontaneous generation [1, 27, 29, 35, 104]. Charlton Bastian (1837-1915) was not convinced that germs were transferred through the atmosphere, and he opposed the theory that diseases are caused by parasites. His experiments showed that some germs may be much more thermoresistant than had been previously supposed. The practice of heating liquids to 115-120°C for sterilization was introduced by Pasteur and Chamberland. Chamberland developed the autoclave and in 1884, a filter made of porous porcelain to remove all microbes from water [27, 35].

John Tyndall (1820-1893), a great experimenter and ingenious thinker, was an opponent of spontaneous generation. He developed a method to make particles in air visible using light scattering and compared the infectiousness of the particle density in air with the growth density in the culture vessels in his apparatus. He observed that the ‘power of air’ to develop life in sterile media paralleled its capacity to scatter light. Air from which all particles were sedimented was shown to be sterile [118]. He concluded that air is a carrier of germs. Tyndall observed, as W. Roberts [119], F. Cohn [76, 82], and Eidam [120] did, that neutralized hay infusion withstands long-term boiling. He concluded that bacteria have phases of development – one phase is relatively thermolabile and is destroyed by heating at 100°C in 5 min, whereas the other phase, which was regarded as the germ of the bacterium, is thermoresistant to boiling temperatures [121]. F. Cohn discovered in the same year that the heat-resistant phase in the development of the hay bacillus is the endospore (Fig. 1). He described the whole life cycle of Bacillus subtilis and the formation and germination of endospores by detailed microscopy studies [82]. On the basis of his results, Tyndall elaborated the important method of fractional sterilization, known today as tyndallization [121]. By this method, vegetative and heat-sensitive stages of bacteria are killed by boiling of the suspension. After a certain period, which is necessary for the heat-resistant germ to pass into a heat-sensitive state, the infusion was boiled a second time. Tyndall determined the time for the interval and the number of repetitions necessary for complete sterilization. The results of Cohn and Tyndall explained many of the controversial results of the advocates and opponents of the doctrine of spontaneous generation, especially the observation that hay infusion, which very often contains heat-resistant spores, resists boiling.

If infusoria are not generated spontaneously from organic matter, how are they originated and propagated? After publication of Darwin’s The Origin of Species in 1859 and the increasing evidence of earlier periods of life on earth, the concept that organisms share a common origin and subsequently diverged through time was slowly adopted by scientists. However, Darwinism revived the discussion on spontaneous generation, although it was restricted to the question of the beginning of life and the origin of microorganisms. In France, Darwinian evolution was regarded as a doctrine allied with the forces that threatened church and state [104]. Ray Lankester in England believed that abiogenesis was a necessary and integral part of the universal evolution theory [27]. The discussion on how the archetype of organisms originated on Earth is still open.

Ernst Haeckel, one of the early propagandists of Darwin’s theory, constructed various phylogenetic trees of organisms [31]. Phylogeny, a term coined by Haeckel, is the attempt to reconstruct the evolutionary history of life. Haeckel divided the organisms into three main groups: animals, plants, and protists. Since the knowledge about bacteria at this time was very meager, Haeckel included them in the collective term infusoria. Cohn, who likewise was convinced of the theory of evolution, speculated that the first living germs arrived from other planets and that all organisms evolved from these primitive organisms – new heritable varieties originated and were separated in specific habitats by natural selection. He proposed that the phycochromaceen were early inhabitants of the Earth because of their ability to adapt to extreme habitats, their simple way of reproduction, and the fossil records [76, 79-81, 84, 85].

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Over much of the next century, biologists’ interest in phylogeny was minimal, but it was rekindled with the accumulation of new phylogenetic data from the field of molecular biology. The development of DNA and RNA sequence technology and of mathematical methods to compare conserved sequences in rRNA and proteins revolutionized evolutionary and phylogenetic biology, especially of bacteria, which developed from an area receiving little attention to one playing a central role in the phylogenetic tree first proposed by Carl Woese and coworkers [99, 100]. The concept of the prokaryotes and the eukaryotes as two groups of organisms, each of which comprises a large evolutionary diversification [99], was largely confirmed and later extended to a tripartite classification of Bacteria, Archaea, and Eukarya [99, 100]. The concept of Cohn [76, 84, 86] that cyanophyceae (now cyanobacteria) are related to bacteria was supported by the concept of the prokaryotic cell [98]. Cyanobacteria were recognized as one of the nearly 20 main lines of descent in the domain of Bacteria [101]. The phylum cyanobacteria and the chloroplasts, which were proposed to derive from endosymbiotic cyanobacteria, have a complex phylogenetic structure [122]. The three groups of organisms – eubacteria, archaebacteria, and eukaryotes – are now generally accepted as major phylogenetic branches of the tree of life. Woese and Fox [99] proposed the progenote as a primitive hypothetical ancestor of prokaryotes and eukaryotes. However, the unraveling of the true evolutionary relationships of the three kingdoms remains a matter of speculation and further studies [123-126].

Archaebacteria have, besides their own typical features, markers which are characteristic of eukaryotes or eubacteria [103, 126, 127]. It has been discussed whether they are polyphyletic and relatives of Gram-positive bacteria [103]. Some physiological attributes, e.g. molecules of the photosynthetic apparatus, are distributed throughout taxa that are phylogenetically not closely related. However, the photosynthetic apparatus seems to have evolved only once [128-130]; the genetic information for their components possibly spread by lateral transfer to different phylogenetic groups. Anoxygenic photosynthesis first developed about 3.5 Gyr ago; oxygenic photosynthesis dominated about 2 Gyr ago [128, 129]. For recent literature on the evolution of microorganisms, see [103, 126, 131-135].

7 The concepts of biological diversity of bacteria

The end products of metabolic processes, such as alcohol or lactic acid, have been known since ancient times, but they were not traced back to the activities of microbes. In the 19th century, the understanding increased that not undefined vital forces, but organisms can metabolize organic and inorganic substrates. This insight was based mainly on the progress in chemical analysis and the observation under the microscope of microorganisms in fermenting, putrefying, and infectious organic material, and was also supported by an increase in rational experimental analysis in biology.

7.1 The organismic and chemical theories of fermentation

For a long time, chemists dominated who proposed that fermentation is caused by a spontaneous internal transformation of organic material ([29], p. 23) or by a ‘ferment’ (‘Gährungsstoff’) [136, 137]. Lavoisier quantitatively studied alcoholic fermentation. He concluded that the sugar was split into an oxidized portion (carbon dioxide) and a reduced portion (alcohol) [138]. Gay-Lussac observed that oxygen is necessary to start fermentation, but not for its continuation [139]. The hypothesis that fermentation, putrefaction, and contagiousness are the result of some spontaneous chemical change in the organic matter or tissues (microzymas) and that microorganisms are just a product but not the causative agent of disease and fermentation [35] was proposed by Liebig [136, 137] and Béchamp [140].

Yeast was considered by Berzelius as a catalyst, like platinum, which was not transformed during fermentation. Liebig postulated that the ‘ferment’ is produced from ‘Kleber’ (gluten) by oxidation with the oxygen from water [136, 137]. G.V.M. Fabbroni [141] suggested that the decomposition of sugar during fermentation is caused, in absence of oxygen, by a vegetative-albumenoid substance.

Caniard de la Tour (1777-1859), Theodor A.H. Schwann (1810-1882) and Traugott Kützing (1807-1893) were the first to propose independently of each other that alcoholic fermentation is a biological process and that yeast is a reproducing, living thing (sugar fungus, Saccharomyces) [142-144]. Schwann and Kützing described yeast in detail. Liebig and Wöhler anonymously published a persiflage of the theory of fermentation caused by living cells [Annalen der Pharmazie 24 (1839) 100]. In a serious article, Liebig [137] described fermentation, putrefaction, and decomposition as processes attributed to the instability of certain substances, which are able to communicate their instability to other substances in succession. He called these unstable, nitrogen-containing substances ‘ferment’, which he believed arose as a modification of a vegetable saccharine solution exposed to air; the fermentation continued in the absence of air. The chemist Mitcherlich concluded from his own observation that yeast is essential for fermentation, but acts by contact, following the catalytic theory of Berzelius [145].

Louis Pasteur (1822-1885) took a great step forward in research on fermentation by studying not only the substrate and the products of growth and fermentation, but also the organisms in the fermentation broth. He investigated the formation of lactic, butyric, acetic, and tartaric acids and of alcohol [64, 114-117] and observed anaerobic life by butyric acid fermentation [64]. In the book Etudes sur la Bière[116], Pasteur summarized and extended his work on fermentation and life without oxygen. The modern view that yeast might ferment sugar through the production of a soluble ferment or enzyme was proposed as early as 1858 by Moritz Traube [146]. Finally in 1897, Eduard Buchner (1860-1907) isolated ‘zymase’ from yeast juice which catalyzed the formation of alcohol from sugar [147]. He opened the door to the fruitful studies of the biochemistry of enzymes, cofactors, and enzymatic processes in the 20th century.

This short overview on the history of fermentation shows that the concepts of catalysis and species-specific metabolic capacities of microorganisms, experimentally proved by new analytical and microbiological methods, paved the way for modern research of this field. The multitude of theories, their controversial discussion, and new experimental approaches accelerated the progress.

7.2 Autotrophy, chemolithotrophy, and phototrophy

Autotrophy, i.e. growth of organisms with carbon dioxide as the only carbon source, was discovered in green plants by Jan Ingenhousz (1730-1799) and Nicolas Théodore de Saussure (1767-1845) [148-150]. The term autotrophy was introduced by Wilhelm Pfeffer [7]. Ingenhousz showed that plants in the presence of light absorb carbon dioxide and liberate oxygen and that the CO2 is used for nutrition [148, 149]. Saussure determined quantitatively the increase of the plant dry weight and the decrease of CO2 during the day in correlation with the light intensity, and he observed that during the night, oxygen is consumed and CO2 is released [150]. Liebig, Boussinggault, and Sachs studied the nutritional physiology of plants grown in a pure mineral solution [19, 151]. The term photosynthesis for the light-induced assimilation of CO2 was coined late in the 19th century. Research on oxygenic (oxygen-producing) photosynthesis increased very slowly, yielding some important results in the middle of the last century (e.g. chloroplasts as the site of photosynthesis, and starch as the product [151]), and culminated in the 1960s with the discovery of the CO2-fixation cycle, the water-splitting system in photosystem II, and the generation of the proton-motive force by light-driven electron transport [152]. After the discovery of photosynthetic CO2 fixation, it was believed that oxygen is generated by the splitting of CO2; the origin of oxygen from water was not discovered before 1941 [153].

Autotrophy and photosynthesis were discovered much later in bacteria than in plants and algae. For many decades, CO2 fixation was assumed to be restricted to plants [76]. Pigmented, bacteriochlorophyll (bacteriopurpurin)-containing bacteria have been described since the 1830s by Cohn, Ehrenberg, Esmarch, Perty, Warming, Winogradsky, Zopf and others [38, 76, 81, 92, 154-157]. Wilhelm Engelmann (1843-1909) investigated the influence of the quantity and quality of light on the movement (photokinesis) and phototaxis of these bacteria [155-157]. He was the first to conclude from growth experiments with these bacteria under anoxic conditions in the light that the ‘purple schizomycetes’ assimilate CO2 like the green plants and that they are able to transform the absorbed light energy into chemical energy [157]. Twenty years passed before the anoxygenic type of bacterial photosynthesis was confirmed by Molisch [154]. Engelmann was not sure because he had contradictory results and was possibly convinced that oxygen liberation is connected with CO2 assimilation.

In 1877, T. Schloesing and A. Müntz observed the bacterial oxidation of ammonia to nitrite and nitrate. R. Warington found that this is a two-step process. Nikolaevitch Winogradsky (1856-1953) isolated the nitrifying bacteria and discovered that the oxidation of ammonia to nitrite by Nitrosomonas and the oxidation of nitrite to nitrate by Nitrobacter yielded free chemical energy, a process which has been named chemolithotrophy [95-97]. He also observed that these bacteria can grow with CO2 as the only carbon source. Thus, these bacteria are chemolithoautotrophs.

Cohn described the presence of hydrogen sulfide in stagnant water bodies and the formation and degradation of sulfur droplets in Beggiatoa (Fig. 2). Erroneously he concluded that Beggiatoa is responsible for the synthesis of H2S [66]. The oxidation of hydrogen sulfide to sulfur and sulfuric acid under microaerophilic conditions in Beggiatoa was demonstrated by Winogradsky [158]. The reduction of sulfate to sulfide by Bacterium desulfuricans was first described by M. Beijerinck [159].

In 1885, H. Hellriegel (1831-1895) and H. Wilfarth discovered the fixation of dinitrogen in root nodules of leguminous plants, and they showed that the combined nitrogen compounds formed in the nodules were supplied to the plants. The causative bacteria were isolated and described by Beijerinck [160]. The reduction of N2 to ammonia by the free-living, aerobic bacterium Azotobacter chroococcum was shown by Beijerinck and van Delden [161, 162], and dinitrogen fixation by the anaerobic bacterium Clostridium pasteurianum was discovered by Winogradsky [163]. Dinitrogen fixation by phototrophic bacteria was detected in cyanobacteria by G.E. Fogg in 1942 and in purple bacteria by H. Gest in 1950. These important results and the progress in the analysis of fermentation and oxidation/reduction processes led to modern concepts of metabolism in the 20th century, e.g. the unifying theory of microbial metabolism proposed by Albert J. Kluyver [164, 165].

7.3 Putrefaction and pathogenicity of bacteria, and immunology

Putrefaction was always observed as a process of decay of organic material, a source of unpleasant odor, and a possible cause of disease in man and animals. The exact nature of the putrid process was, however, for a long time a matter of speculation because no experimental approach was available [1, 14, 35, 36, 166]. In the second part of the 19th century, growth experiments using selected and defined media with cultures of microorganisms enriched in one species revealed that microorganisms have specific, inheritable features for the production of pigments or fermentation products or for the oxidation of inorganic compounds. Putrefaction was traced back to the activity of bacteria able to decompose nitrogen-containing organic material. The standpoint of Liebig, that putrefaction is a rearrangement of molecules, was refuted [76, 116]. Four possibilities for the mechanism of putrefaction were discussed by Cohn [76]: (i) bacteria assimilate proteins and transform them into their own cell material; (ii) bacteria produce and excrete a ferment-like compound which solubilizes and decomposes protein (like barley grains, which produce diastase, which splits starch into sugar); and (iii and iv) bacteria oxidize or reduce protein enzymatically with oxidizing or reducing ferments. It was concluded that pigmented bacteria or other bacteria split protein molecules after uptake directly or by excreted ferments into ammonia and other compounds and assimilate ammonia as nitrogen source [76].

7.3.1 Bacteria as contagion

The process of putridity was more and more associated with the idea of sepsis as a cause of septicemia, pyemia, and putrid infection. B. Gaspard administered putrid material to experimental animals and observed the development of symptoms. The nature of pyemia and septicemia, however, remained a mystery [35]. The pathological effects of putrid infections on the cellular level were described by Virchow [167]. He and other scientists studied the effect of dose on the symptoms of septic shock, intoxication, abscesses, and cytopathic modifications of tissues [36, 166-168]. Koch [14, 69] investigated the histology of infected tissues, and he examined with a microscope fitted with an oil-immersion lens system and an Abbe condenser the germs in slide preparations stained with aniline colors. He stressed that each type of infection resulted in a characteristic histological picture caused by the microorganisms specific for the illness; the bacterial causative agent had to be isolated from the tissue [14]. Several scientists, such as E.v. Bergmann (1868), Hiller (1879), and Blumberg (1885), were opponents of the germ theory of putrefaction and proposed that putrefaction is caused by toxic compounds [35, 36]. The observation of vibriones (Treponema pallidum) in syphilitic pus and Trichomonas vaginalis in the vagina by Donné, the detection of the anthrax bacillus by C.J. Davaine [169], the description of a mold, called Botrytis bassiana (Beauveria) as a causative agent of a silkworm disease by A.M. Bassi [170], and other observations motivated Jacob Henle (1840) to reactivate the germ theory of disease and to study infectious diseases [62]. This was at about the same time that Pasteur’s studies revealed specific microorganisms as the causative agents of different fermentations.

The numerous cholera epidemics, the observations of Davaine on anthrax, of Traube (1864) and E.K. Klebs (1869) on urethritis and nephritis, of Rindfleisch (1869) on affections in the heart muscle, the demonstration of Borrelia recurrentis only during an attack of fever in the blood of patients suffering from relapsing fever by O. Obermeier [171], and other observations on infected tissues [172] stimulated E. Hallier (1831-1904) to isolate and cultivate germs from infected tissues in his culture glass [173, 174]. Unfortunately he interpreted the germs, isolated from humans and animals infected by cholera, typhus, gonorrhea, diphtheria, and pox virus, as forms of fungi (polymorphism). This incorrect interpretation was rejected by the mycologists de Bary and Brefeld and bacteriologists such as Cohn, Rindfleisch, and Burdon-Sanderson [35, 36]. In spite of the inconsistent results of the numerous observations, the view that bacteria and other microorganisms may be the causative agents of the diseases received increasing consent ([36], pp. 86-103).

The crucial experiments showing that one distinctive species causes a specific infectious disease were performed by Robert Koch (1843-1910). The decision to select anthrax for the first series of experiments was fortunate because the infected tissues contain numerous bacilli (Bacillus anthracis). Koch isolated the bacteria from an animal that died of anthrax and set up cultures in a moist chamber, where the growth, division, and sporulation of the bacilli were observed with the microscope and documented by microphotography, which was developed by Koch [12]. In the decisive experiment, it was shown that B. anthracis, isolated from the diseased animal, and not Bacillus subtilis, caused anthrax in mice. Koch wrote to Cohn, who was at this time an internationally recognized authority in bacteriology, that he would like to demonstrate his results in Cohn’s laboratory. The demonstration of his decisive results in Cohn’s laboratory in Breslau in the presence of Julius Cohnheim and Carl Weigert, both pathologists in Breslau, was an important step in the progress of infectious biology and the first proof that distinct bacterial species can cause a disease with typical symptoms. The results were published [94] after Cohn’s article on the sporulation of B. subtilis[82] in the journal Beiträge zur Biologie der Pflanzen, founded by Cohn. The experimental proof of infectious disease, later described as Koch’s postulates, was the beginning of modern medical microbiology [13, 35, 36]. The postulates comprise the isolation of the microorganism from the infected tissues, the growth of the microorganism in pure culture using the plate and slide technique [69, 175], the improved methods of observation using microscopy, staining and documentation of the bacteria [10, 12, 175], the achievement of the typical symptoms of the disease by inoculation of the isolated bacteria into a sensitive host [176-178], and the isolation of the same microorganism from the newly infected host. The first success was followed by the discovery of Mycobacterium tuberculosis as the causative agent of tuberculosis [177, 178]; of Corynebacterium diphtheriae, which causes the toxigenic disease diphtheria [179]; and of Vibrio cholerae, which causes cholera [180]. A. Ogston, using Koch’s technique to isolate and determine the number of micrococci in pus, cultivated cocci in glass cells with Cohn’s or Pasteur’s fluid under oxic or anoxic conditions and concluded that Streptococcus (Rosenbach) and Staphylococcus cause inflammation and suppuration [181, 182]. Rosenbach [172] subdivided the genus Staphylococcus into species. In summary, the development of several new techniques and the concept that specific infectious diseases are caused by distinct species of bacteria having inheritable virulence factors were the prerequisite for placing research on the etiology of infectious diseases on a valid scientific ground.

7.3.2 Control of infectious diseases

While Koch and his coworkers and disciples were concentrating on the isolation and characterization of parasitic and saprophytic bacteria, the French school under the guidance of Pasteur was directed toward the prevention of infectious diseases. By repeated passages of pathogenic germs on artificial media at high temperatures (42°C), in non-host animals, or in specific tissues, they obtained bacteria with attenuated virulence. These attenuated cultures were used to inoculate animals or humans. The vaccinated individuals were shown to be resistant to the virulent strains of fowl cholera, anthrax, swine erysipelas, or rabies [183-186].

7.3.3 Disinfection

The observation that carbolic acid inhibits growth of microorganisms and the formation of pus in wounds [187] and the numerous observations that microorganisms cause fermentation, putrefaction, and infections stimulated Joseph Lister (1827-1912) to develop the antiseptic system [188, 189]. He worked out procedures for treating wounds with phenolic compounds that were generally accepted and introduced into the medical practice after a long period of uncertainty. Koch and others tested many other disinfectants [190, 191].

7.3.4 Immunology

From studies on the interactions of warm-blooded bodies with inoculated parasites and the successful fight against infectious diseases, research in the large field of immunology was initiated and the theories of humoral and cellular immunity were developed [29, 35]. E. Metchnikoff (1845-1916) studied phagocytosis [192], and E. von Behring (1854-1917) and S. Kitasato (1852-1931) detected the antitoxins against infections of Clostridium tetani and Corynebacterium diphtheriae[193]. Paul Ehrlich (1854-1915) published important articles on toxins and antitoxins and their standardization and on the specificity of antibodies against antigens [194, 195]. The extreme complexity of the immune system was revealed at the end of the 19th century.

8 The achievements of Ferdinand Cohn

Ferdinand Cohn was born on January 24, 1828 in Breslau, now Wroclaw, into a Jewish family. His life-long interest in history and the classical languages Latin and Greek was born during his education at the Maria Magdalena Gymnasium (Fig. 3). After the final examination at this Gymnasium he began his studies of natural sciences with the main subject botany in Breslau in 1844 (Fig. 4). Cohn continued his studies from 1846 to 1849 in Berlin because his application for admission to the doctoral examination at the university in Breslau was refused because of his Jewish faith. He received his doctoral degree in Berlin on November 13, 1847 at the age of 19. In 1849, Cohn returned to Breslau full of ideas for studying developmental cell biology, especially of lower plants and microorganisms by means of microscopy methods. He completed his Habilitation (second dissertation) in October 1850, became a lecturer (außerplanmäßiger Professor) on April 2, 1857, and an associate professor on July 30, 1859. He married Pauline Reichenbach in 1866, and was appointed full professor on April 17, 1872 (Fig. 5). In 1866, he founded the Institute of Plant Physiology and established a research group (Fig. 6). A new building for plant physiology, a herbarium, and a museum of botany were constructed in the botanical garden of the university and were opened in 1888 (Figs. 7 and 8). Cohn died on June 25, 1898 (Fig. 9). The details of his career have been described recently [79, 196].

Cohn contributed to a broad field of topics in biology. His reserve in self-representation and his modesty may be the reason why his name is at the present time much less known than that of Koch and Pasteur. Cohn’s major field during his studies in Breslau and Berlin was botany. In Berlin he received a decisive intellectual stimulus for his subsequent research. His studies on plant cells and on the development and sexuality of algae and fungi established his early distinction in the scientific community [18, 20-25, 56, 57, 65]. The leading role of Cohn in the evolution of the principles of modern bacterial taxonomy was described in Sections 4.1 and 5 [66, 76, 79, 81, 84-86] and his comprehensive studies of bacterial physiology were detailed in Section 7 [65, 66, 76, 82, 85]. In addition, Cohn was an important promoter of applied microbiology. He gave lectures in agricultural botany, advised farmers on the diagnosis and treatment of plant diseases caused by fungal infections, became a pioneer in the analysis of water as one possible source of infectious diseases, and described in detail the damage to trees by hurricanes and lightning [80, 197-200].

In 1875 on the 50th anniversary of the doctorate of his mentor Professor Göppert, Cohn initiated the work on the flora of cryptogams in Silesia and then published the work in several volumes between 1877 and 1908 [201]. In the ‘Schlesische Gesellschaft für Vaterländische Kultur’ (the Silesian Society of National Culture), a type of academy in which he served as the secretary of the botanical section for more than 30 years, it was decided that the developmental stages of approximately 90 plants should be observed and documented by volunteers throughout the year at different places in Silesia. Cohn reported and coordinated the registered changes in the vegetation for many years [202]. Cohn published important contributions on the movement of plants induced by external stimuli (light, chemical, and mechanical) [203-208].

Cohn’s comprehensive knowledge in many fields of biology was combined with a deep interest in history and art. Cohn felt obliged to mediate knowledge of natural sciences to a wide audience. He was convinced that education in natural sciences was as important as training in cultural sciences and that scientific thoughts are important not only for scientists, but also for the general public to open their minds. He stressed and practised this idea during his entire life. All those who listened to him were impressed by his gifted, clear, rhetorical, and brilliant speech. His popular description of a broad spectrum of botanical knowledge was combined in the collected lectures on plants, which was published in 1882, and in a second, revised edition in 1897 [209]. The article on ‘Plants in the fine arts’ [210] presents a retrospect on the description of plants in art and science during several epochs. In 1856 he gave a lecture in Berlin on the history of gardens.

One of the postulates that Cohn defended during his oral doctoral examination was the need for an Institute of Plant Physiology. During his academic career, he never neglected this goal, but the realization was impeded by many obstacles. By his continuous effort, he was allowed in 1866 to use several empty and dark rooms in the old convent of the university located in the center of the town to set up a laboratory of plant physiology (Fig. 6). By difficult and continuous negotiations with the ministers of agriculture and culture, he received a small amount of financial support for the costs of the equipment, lighting, sanitary installation, and the salary of a technician. Very often, Cohn himself laid out the money and was reimbursed after many months [211-213]. Many students and scientists came from abroad to study with him and many of these students later obtained leading positions. The inadequacy of rooms for research and teaching and the need for a museum of botany finally led, after many years of planning and discussion, to a successful proposal for a new building, which was opened with a ceremony on April 29, 1888. The new building, located in the botanical garden, contained the herbarium and the museum, a lecture room, the Institute of Plant Physiology with laboratories and the library, the office for the director of the botanical garden, and apartments for employees [213]. The building is still in use (Fig. 7).

From the beginning of his career, Cohn communicated with many of his contemporaries by letter and by personal contact during meetings and private journeys [214]. During the last decade of his life, when he was no longer at the forefront of science, he published articles on historical aspects and overview articles, e.g. on Tabaschir or mandragora [215], the history of botany and botanical gardens [216-218], Caspar Schwenckfeld [219], and Laurentius Scholz von Rosenau [220]. Cohn was honored by numerous distinctions from academies, societies, universities, and his home town Breslau. The German Society of Hygiene and Microbiology awarded a Ferdinand Cohn medal in the 1980s. His great personality and his important work have been illuminated in several articles [35, 79, 196, 212, 221-227].

Cohn was a personality molded by an education in classical art. His Jewish faith and his modesty led him to avoid political activities, although as a student he participated actively in the 1848 revolution in Berlin. His sustained importance in biology was in two fields: the sexuality and development of lower plants and the concept that bacteria are organisms with distinct, heritable characteristics. He proposed principles of bacterial taxonomy, knowing that at his time the methodical means to elaborate a modern taxonomy on a phylogenetic basis were not available. Cohn discovered the development and heat resistance of bacterial endospores.

References

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