201: Microbiology UNIT –I 1. Beginning of Microbiology, milestones in the development of microbiology, spontaneous generation, Microbial Ecosystem, Microbial world, Branches of Microbiology, Application of microbiology. 2. Methods in Microbiology: Sterilization, Culture Media, Pure culture technique, enrichment culture technique, Microbial staining methods, Maintenance and preservation of Microorganisms, Culture collection centers. 3. Microbial growth: Growth curve, measurement of growth, growth yields, synchronous growth, continuous culture, growth as affected by environmental factors such as temperature, acidity, alkalinity, water availability and oxygen. 4. Microbial evolution, systematics and taxonomy: Evolution of earth’s earliest life forms, primitive organisms, their metabolic strategies and their molecular coding, New approaches to bacterial taxonomy, nomenclature, Bergey’s manual, Ribotyping. NOTES Discovery of Microscope: The fascinating microbial world would have remained unknown had the microscope not been invented. It was Roger Bacon (1267) who developed a lens for the first time. Jansen and Jansen (1590), about 300 years later first produced a crude type of microscope by placing two lenses together without any provision for focusing’ Galileo Galilei (1610) prepared a microscope with a focusing device called ‘occiale’. Till then, the name ‘microscope’ had not been in use and it was first proposed by Faber (or Fabri) in 1625. However, the advent of such optical lens systems did not reveal the existence of microorganisms. It was not until the mid-17th century when further development of the optical lens systems to definite microscope permitted the visualization of microorganisms that the great diversity of the microbial world began to be recognized. Robert Hooke (1635-1703) made and used a compound microscope in the 1660s and described his fascinating explorations of the newly discovered universe of microscopic creatures in his classic “Micrographia” (1665). Although Hooke’s highest magnifications were possibly enough to reveal bacteria, he apparently could not see them probably because he studied mainly opaque objects in the dry state by reflected light, conditions that are not optimal for observing bacteria. However, his pictures of “white moulds” (probably a Mucor species) are very informative and accurate (Fig. 1.1). 2. Discovery of Microbial Life: The exact beginning of the knowledge about the existence of microorganisms can be traced back only to the latter part of the seventeenth century when Antony van Leeuwenhoek (1677) first recorded observations of microorganisms (bacteria, yeasts, and protozoa) seen in water, faeces, teeth scrappings etc. under his own microscopes (Fig. 1.2) which were not compound. Leeuwenhoek (1632-1723) was basically a cloth maker and tailor by trade, was also a surveyor and the official wine taster of Defft, Holland and his interest in microscopes was probably related to the use of magnifying glasses to examine fabrics. He transmitted his findings in a series of more than two hundred letters to the Royal Society of London during his lifetime. He described such tiny creatures as “dierkens” or “animalcula viva” which were translated in English as “animalcules” by the Royal Society. Leeuwenhoek was later elected a fellow of the Royal Society. Although there are reports of works on microorganisms, O.F. Muller gave first classification of bacterial microbes in 1773 and 1788, and coined the terms “Vibrio and “Monas” for certain forms; Ehrenberg established a new genus ‘Bacterium’ in 1829. Leeuwenhoek’s animalcules took two centuries to cause any spurt among the scientists when their importance was realized in different areas of human affairs. 3. Abiogenesis Versus Biogenesis (Microbes and the Origin of Life): 1. Spontaneous Generation Doctrine (or Abiogenesis): Men of ancient times (Thales, 624-548 B.C.; Anaximander, 611-547 B.C.; Anaximenes, 588-524 B.C.; Empedocles, 504-433 B.C.; Aristotle, 384-322 B.C.; Epicurus, 341-270 B.C.; and Socretius 99-55 B.C.) knew nothing of microorganisms, of evolution, or of the fact that only living things could beget living things. They believed that all living organisms could spring forth spontaneously from non-living matter. This belief has been referred to as Doctrine of Spontaneous Generation or Abiogenesis (Gr. a = not; bios = life; genesis = origin). They believed that frogs, snakes and mice could be born of moist soil, that flies could emerge from manure, and that maggots could arise from decaying corpses. The idea of spontaneous generation was supported even 2000 years later. Van Helmont (1577-1644) devised a method for manufacturing mice. He recommended putting some wheat grains with soiled linen and cheese into an appropriate receptacle and leaving it undisturbed for a time in an attic or stable. Mice would then appear. However, the idea of spontaneous generation continued until the mid-19th century with great oppositions against it. 2. Controversy over Spontaneous Generation: Actually, it was the discovery of microorganisms and improvements in microscopy that enabled scientists to think seriously about the origin of life. Francesco Redi (1626-1679), an Italian physician, demonstrated during mid-17th century by simple experiments (Fig. 1.3) that spontaneous generation (abiogenesis) does not exist. He took rotting meat pieces and placed them in jars. He sealed some of these jars tightly and left others open. In a few days, maggots appeared in open jars in which the flies went freely in and out and laid their eggs on meat. Contrary to it, the sealed jars in which the flies could not enter did not show any maggots. From these observations Redi concluded that the maggots arise from the eggs laid down by the parent flies and that the maggots cannot appear spontaneously. Still, the supporters of abiogenesis did not agree with Redi and argued that the free air, which was considered as “vital force” necessary for spontaneous origin of life, was not allowed to reach the meat placed in sealed jars. So Redi set up new set of experiment in which he covered jars with fine muslin cloth or gauze instead of sealing them tightly and thus allowed free air to go in and out of the jars. Even after doing so the maggots appeared only in those jars in which flies were allowed free to go in and lay their eggs on the meat. Even after Reid’s convincing demonstration, abiogenesis versus biogenesis controversy continued. John Needham (1745) advocated that even after he heated chicken broth and corn infusions (nutrient fluids) before pouring them into covered flasks, the cooled solutions showed existence of tiny organisms in them and thus he claimed that the organisms originated spontaneously from the nutrient fluids. We shall see later that this result was due to insufficient heating which failed to kill heat-resistant forms of bacteria containing endospores. But nothing was known about endospores at that time. In the year 1765, twenty years later Lazzaro Spallanzani demonstrated that nutrient fluids of Needham did not contain microorganisms when they were subjected to prolong heating after being sealed in flasks. He explained that the microorganisms from air probably had entered Needham’s solutions after they were boiled. Needham responded to it and said that the free air, the “vital force”, present inside Spallanzani’s scaled flasks had been destroyed by heating and, therefore, microorganisms did not appear in nutrient fluids in absence of the “vital force”. 3. End of the Debate: Irritated by continuous advocacy in favour of spontaneous generation even by nineteenth century scientists, Louis Pasteur (1861) conducted series of experiments to prove that if the solutions are made microbe-free by boiling and they are provided with microbe-free air (the -vital force” for spontaneous generation), they do not show any sign of spontaneous origin of microbial life in them. In his swan-necked flask experiment (Fig. 1.4), he took various type of broths (yeast water, sugared yeast water, urine, sugar beet juice etc.) in long-naked flasks and, then, softened the neck of the flasks under a flame and drew it out in the shape of ‘S’ looking like the neck of the swan. The broths of these flasks were boiled until they steamed through the necks, and then cooled. The broths so treated in the flasks did not decay, and there were no signs of microorganisms in them after days, weeks and even months though they were open to free-air. Pasteur’s unique swan-necks of the flasks trapped air-borne microorganisms before they could reach the broth and flourish in it. The broths in the flasks open to free air but free of microbes for very long periods, therefore, definitely discredited the doctrine of spontaneous generation. Despite Pasteur’s successful demonstrations against spontaneous generation, attempts to repeat his experiments occasionally failed because, after some time, existence of microbes was evident in some broths of swan-necked flasks. This created doubt in the minds of many. But, this problem was soon solved by John Tyndall, an English physicist, in the year 1877. He explained that bacteria exist in two forms: Heat-labile forms (thermolabile) which could be killed by exposure to high temperatures, and heat-resistant forms which could not be killed by continuous boiling of the broth and, after the broth has cooled, they resulted in microbial growth in such broths. He further stated that if such broths are subjected to intermittent boiling (discontinuous boiling) on successive occasions, a process now popular as tyndallization, the heat-resistant forms of bacteria will be killed and the broths become completely free of them, and do not show any microbial growth. It so happens because the first boiling kills vegetative cells of bacteria but endospores remain as such. The endospores now germinate in cooled broth and produce new bacterial cells which are killed during further boiling and so on. In this way, Tyndall validated Pasteur’s results and helped ending the debate on abiogenesis versus biogenesis.