GENERAL MICROBIOLOGY Microbial World, History and Development of Microbiology, Scope of Microbiology Dr. (Mrs) S. Sharma Professor Department of Microbiology CCS Haryana Agricultural University Hisar-125001 and Dr. Neeraj Dilbaghi Reader Department of Biotechnology Guru Jambheshwar University of Science & Technology Hisar-125001 01-May-2006 (Revised 12-Dec-2006) CONTENTS Introduction Historical Developments Leeuwenhoek’s Findings Biogenesis versus Abiogenesis Contributions of Pasteur Contributions of Robert Koch and Germ Theory of Disease
Rise of Medical Microbiology Microorganisms as Geochemical Agents Microbiology in the Twentieth Century Activities of microorganisms important in our daily life Scope of Microbiology Some recent applications of bacteria Keywords Microorganisms, microscope, fermentation, disease Introduction Microbiology, the study of microscopic organisms, derived its name from three Greek words: mikros (“small”), bios (“life”), and logos (“science”). Taken together they mean the study of microorganisms which are very small and cannot be seen by unaided eye.
If an object has a diameter 0. 1 mm or less, eye cannot see it and very little details can be seen in an object having diameter 1 mm. So roughly speaking organisms having diameter 1 mm or less are called microorganisms and are studied in Microbiology. Although microorganisms are ancient by many standards, microbiology itself is a comparatively new science. The existence of microorganisms was unknown until the discovery of Microscope. Microscope is an optical instrument which can magnify small objects which cannot be seen by naked eye.
Microscopes were invented in the beginning of 17th century. Early Microscopes were of two types; Simple Microscope, with a single lens of very short focal length and Compound Microscope, with two double convex lens system including ocular and objective lens with higher magnifying power. Most of the epoch making original discoveries about microorganisms was all made using simple and compound microscopes. Characteristics of microorganisms 1. Their size is very small. 2. There is no cellular differentiation.
They are unicellular and one cell is capable of performing all the functions. Some microorganisms are multicellular with little or no cellular differentiation. 3. Microorganisms are present everywhere on the bodies of animals and humans, on plant surfaces, in the air, water, dust, soil, and even inside the intestinal canal of all insects, birds, animals and human beings. Taxonomic Groups Microorganisms have wide taxonomic distribution and include organisms such as protozoa, algae, fungi, bacteria and virus.
The schematic illustrations of different microorganisms are shown in Figure S. 1. Protozoa are unicellular eukaryotic organisms, motile having cilia, flagella and pseudopodia, saprophytic or parasitic. They are generally present in soil, water and marshy places and their size varies from 5-200 µm. They are animal-like in that they ingest particulate food, lack a rigid cell wall, do not contain chlorophyll. The study of protozoa is known as protozoalogy. They are differentiated on the basis of morphological, nutritional and physiological characteristics.
Their role in nature is varied, but the best known protozoa are the few that cause disease in human beings and animals, such as malaria in humans. Some protozoa are beneficial, such as those found in stomach of cattle, sheep and termites that help digest food. Algae are relatively simple organisms, their size varies from 1 µm to several feet. They are considered plant-like because they contain the green pigment chlorophyll, carry out photosynthesis, and have rigid cell walls. They are unicellular to multicellular and either motile or nonmotile. The study of algae is known as Algology or Phycology.
These organisms are autotrophic and are found most commonly in aquatic environments or in damp soil. They cause problems by clogging water pipes, releasing toxic chemicals into water bodies, or growing in 2swimming pools. But extracts of some species have commercial uses: as emulsifiers for foods such as ice-creams; as a source of agar used as solidifying agent in microbial medias and as antiinflammatory drugs for ulcer treatment. Figure S. 1. Schemetic illustrations of different microorganisms. [A] Bacillus cereus, [B] Staphylococcus aureus, [C] Saccharomyces cerevisiae, [D] E. oli, [E] Listeria monocytogenes, [F] Red algae, [G] Blue green algae Oscillatoria, [H] Amoeba, [I] Hepatitis virus, [J] Euglena A B C D F E G H J I 3Fungi are either saprophytes or parasites. They have eukaryotic cell structure which, like algae, have rigid cell walls. They form characteristic hyphae called mycelium which may be septate, nonseptate or coenocytic. They form fruiting structures called conidia or exospores and endospores. Spores of fungi are always present in air, dust and soil. Multicellular fungi are also called molds while yeast is an important unicellular fungus.
Size range of molds is 2. 0-10 µm and yeast has size varying in the range of 5-10 µm. Molds have considerable value; they are used to produce antibiotics- penicillin, cephalosporin etc, fermented products like soy sauce, tempeh, miso, Roquefort and Camembert cheeses, and many other products. But they are also implicated in various human, animal and plant diseases including athlete’s foot and the moldy spoilage of grains and peanuts. The unicellular yeasts are widely used in Baking industry and for the production of all alcoholic beverages like wine, beer etc.
On the other hand, some yeasts cause food spoilage and diseases such as vaginitis and thrush (an oral infection). Bacteria are unicellular microorganisms. Their size varies from 1-5 µm and have rod, coccus or spiral shape. They have prokaryotic cellular organization and cell division is usually by binary fission. Some bacteria having mycelial morphology are known as Actinomycetes and are very important in production of antibiotics. Bacteria are important in agriculture and play important role in cycle of biological nitrogen fixation.
With respect to food, they are important in fermentations, food spoilage, food poisoning and food preservation. The wide range of industrial products derived from bacteria affect the human society in numerous ways. Their activities are of enormous importance and some are beneficial while others are harmful. The study of bacteria is known as Bacteriology. Viruses are ultra-microscopic, noncellular obligate parasites of plants, animals and bacteria as well as other protists. Their size varies from 0. 015µm -0. 2 µm and shapes from spherical, rod, flexuous to cozohedral.
They can be seen only under an electron microscope. Unlike cells, viruses contain only one type of nucleic acid, either DNA or RNA, which is surrounded by a protein-coat. They lack the cellular components necessary for metabolism or independent reproduction, viruses can mutiply only on living cells. The study of viruses is known as virology. Viruses cause large number of diseases in humans (such as AIDS, common cold, poliomyelitis, SARS, genital herpes, hepatitis etc), plants (tobacco mosaic disease, papaya ring spot disease etc) and foot-and-mouth disease of animals.
In addition, some retroviruses have also been implicated in the growth of some malignant tumors. Historical Developments in Microbiology Some momentous discoveries in science were made by amateurs, rather than by professional scientists. One of such major stalwarts in the history of microbiology a natural scientist, owned his own dry goods store, and was also the official wine taster for the city of Delft in Holland. Antony van Leeuwenhoek (1632 -1723; Figure S. 2A) known as the Father of Microbiology, was a pioneer in the field of Microscopy and used microscopes of his own design and manufacture.
He was a linen merchant who built microscopes as a hobby, had little formal education and knew only Dutch language. He made about 500 optical lenses that could magnify objects 275 times and was an amazing feat. He enjoyed using his microscopes to look at the various things including river water, pepper infusions, saliva feces and more. He communicated his findings to the Royal Society of London in the form of long series of letters which were translated and published in the Proceedings of Royal Society. Leeuwenhoek constructed many microscopes with a single lens which consisted of a spherical lens mounted between two small metal sheets of silver or brass (Figure S. 2B). The specimen was placed on the point of a blunt pin and brought into focus by manipulating two perpendicular and a linear screws. Leeuwenhoek’s Findings Leeuwenhoek had unusual degree of curiosity and observed every object that could be seen through his microscope. He was one of the greatest Innovators driven by curiosity and infinite energy.
In his letter of September 17, 1683 with his drawings about ‘animals’ in the scrapings of teeth he described different types of bacteria and called them ‘animalcules’ (Figure S. 2C). He also made magnificent observations on the microscopic structure of seeds and embryos of plants and some invertebrate animals. He discovered Spermatozoa, RBC and is therefore known as Father of Animal Histology. He described characteristic microflora of human mouth, curd, vinegar and infact discovered all the different types of microorganisms known today including protozoa, algae, yeast and bacteria.
He also emphasized the abundance of these microorganisms besides their great diversity. Figure S. 2 [A] Antony Von Leeuwenhoek. [B] The Leeuwenhoek’s microscope. [C] Leeuwenhoek’s sketches of bacteria from the human mouth. He was elected a fellow of the Royal Society of London (FRS) in 1680. The Royal Netherland Academy of Arts and Science established a ‘Leeuwenhoek Medal’ in his honor in 1877. The ‘Antony van Leeuwenhoek chair’ established in Delft places a strong emphasis on research and appointments are made on the basis of achievements. 5Origin of Leeuwenhoek’s microorganisms
Soon after the discovery of microorganisms by Leeuwenhoek, scientists began to study the origin of these small organisms from the point of view of the two schools of thought; One believed in the Theory of Abiogenesis or the concept of spontaneous generation i. e. living animalcules are formed spontaneously from nonliving matter, while the other supported the theory of biogenesis i. e. they are formed from the ‘seeds’ or ‘germs’ of these animalcules which are always present in the air. It was also believed at that time that many plants and animals can be generated spontaneously under special conditions.
Biogenesis versus Abiogenesis It took several clever experiments, which appear too simple today, and more than hundred years to resolve the controversy. The abiogenesis for plants and animals was disapproved as a result of the experiments by Italian Physician Fransesco Redi in 1665, who showed that maggots developing in putrefying meat are the larval stages of flies and will never appear if the meat is well protected in a vessel with the fine gauge mesh so that flies cannot lay their eggs on meat. In 1745, John Needham took hot boiling mutton gravy (meat infusion) in a flask and closed this flask with a cork.
He found the spoilage of this infusion and observed animalcules in it. He killed and destroyed the living matter by boiling and thus concluded that animalcules arose spontaneously from the meat. In 1769, Lazaro Spallanzani (1729-1799) an Italian naturalist performed a series of experiments and showed that heating can prevent appearance of animalcules in infusion although duration and level of heating required is variable. He was not satisfied with using cork to plug the flask and sealed it hermetically to prevent contact with the air completely.
He found that sealed infusions remained barren for a long time. A tiny crack in the flask can result in development of animalcules and they will not appear unless new air entered the flask to come in contact with the infusion. Spallanzani infact took a series of flasks and gave heat treatments for different interval of times. He could distinguish animalcules of different types, i. e. , Superior or animalcules of higher class which were destroyed by slight heating undoubtedly protozoa and animalcules of lower class which were very minute, and much more heat resistant – the bacteria.
Although the experiments Spallanzani conducted were very good but faulty experiments continued to be performed and evidence gathered in favor of abiogenesis. Moreover, Needham objected to the observations by Spallanzani that there was no growth in the infusions because air which is essential for life had been excluded from his flasks. An interesting practical application of Spallanzani’s observation was done by Francois Appert (1805) for preservation of foods by enclosing them in airtight containers and then heating the containers called ‘Appertization’.
He was thereby able to preserve highly perishable food and the process named canning (called later) was widely used much before its scientific principles were understood. He performed an experiment by first making the air free of microorganisms by passing it through red hot tube. This air which still contained 19. 4 % oxygen, with and without heating was passed through a set of flasks containing boiled infusions, the former remained unaffected. 6Proof of Biogenesis During the period when these experiments were done, a new figure was emerging in science, in the name of Louis Pasteur (1822-1895) in France (Figure S. ). A chemist by training, he emerged as one of the greatest biologists of the 19th century. His contributions are the most significant in the history of science and industry and his work with germs and microorganisms opened new areas of scientific studies. Pasteur was born on 22nd Dec 1822 in the eastern French town of Dole and later became the Dean of the new science faculty at Lille University in 1854. We pay tribute to him as he was a great benefactor of humanity. Figure S. 3: Louis Pasteur in his laboratory
Pasteur first demonstrated through a series of definitive experiments that air contains microscopically observable ‘organized bodies’. He aspirated large quantities of air through a tube which contained a plug of cotton to serve as a filter. He removed the cotton plug and suspended it in a solution of alcohol and ether. When he examined the sediments microscopically he observed the presence of small oval shaped bodies. He later confirmed that when heated air is passed through a boiled infusion no microbial development takes place but when cotton plug is suspended in the heated infusion, microbial growth occurs.
Pasteur repeated his experiment through swan necked and gooseneck flasks (Figure S. 4) so that the germs from air cannot ascend into it. He boiled the broth in it to kill all microorganisms in the neck as well as in the flask. The infusion remained sterile in this flask until the neck of the flask was broken resulting in the growth of microorganisms. Thus he established that development of microorganisms in organic infusions bring about chemical changes. Schroeder and Von Dusch (1850) started the technique of cotton plugging because cotton acts as a filter and traps microorganisms.
One of the traditional arguments against biogenesis was the claim that heat used to sterilize the air or specimens was destroying an essential “vital force”. Those supporting abiogenesis said that, without this force, microorganisms could not spontaneously appear. In response to this argument, an English Physicist John Tyndall (1820-1883, Figure S. 5) conducted experiments in a specially designed box called ‘Tyndall chamber’ to prove that dust carries the germs. He demonstrated that if no dust was present, sterile broth remained sterile for indefinite period.
While doing these experiments, Tyndall (1877) also devised a process for complete sterilization by alternate heating and cooling known as ‘Tyndallization. ’ He found that in some cases even 7boiling the infusion for more than 5 hours was not sufficient to sterilize it and concluded that bacteria have both thermo stable and thermo labile phases. These thermo stable resting bodies were also observed by Ferdinand Cohn in hay bacteria and were called endospores. The experiments by Pasteur and Tyndall finally disapproved the theory of spontaneous generation and promoted the general acceptance of theory of biogenesis.
Figure S. 4: Pasteur’s gooseneck flask Figure S. 5: John Tyndall Other contributions of Pasteur Pasteur’s inventions were based on his work on fermentation (1857 -1876) and had a practical significance. He was called by a winery in Lille in France where they were facing the problem of 8getting a poor product. Careful investigation of the problem by Pasteur led to conclude that alcoholic fermentation was replaced by another type of fermentation which converted sugar to lactic acid. Not only did he find the reason for the problem but also the remedies.
It was his ability to apply and relate discoveries to practical world that made his contributions very significant. The important contributions made by Pasteur are summarized as ; a) Fermentation is a biological process and is brought about by development and activities of microorganisms, b) A typical fermentation can be defined by its principal end product, e. g; lactic, alcoholic and acetic acid fermentation, c) Fermentation is a specific process meaning thereby every fermentation is accompanied by development of a specific type of microorganism which shows physiological specificity with respect to fermentation e. . , alcoholic fermentation by yeast and lactic acid fermentation by lactic acid bacteria. Pasteur also discovered the process known as pasteurization after his name for preservation of wine by sudden heating to 60-700 C for few minutes and then cooling to destroy the harmful organisms. This not only saved the wine industry but the process was also applied later to preserve milk and other liquid foods. Today pasteurization is widely used in fermentation industries, but we are more familiar with it in the dairy industry. Germ theory of Disease
Even before Pasteur proved by experiments the possibility of microorganisms as agents of disease, several careful observers had made strong arguments for the germ theory of disease. A little earlier to this, John Bassi in1836 and M. J. Berkeley in 1845 had shown that silkworm disease and the great Potato Blight of Ireland was caused by fungus. Few years later, J. L. Schonlein showed that certain skin diseases in humans are caused by fungal infections. In 1860 a disease called ‘Pebrine’ was killing large number of silkworms and destroying the silk industry.
Pasteur described that microbes were killing the silkworms and eliminating the worms will wipe out the disease. He also demonstrated that by weakening the disease germs in lab and then infecting the weakend germs into animal or person, the animal developed immunity against that disease. Antiseptic surgery In the 1860’s, an English surgeon named Joseph Lister was searching for a way to prevent microorganisms infecting wounds, as deaths from post surgery infections were frequent and accounted to about 45% of the total deaths.
Lister used dilute solution of phenol/carbolic acid to soak surgical dressings and by performing surgery under a spray of disinfectant to prevent airborne infections (Figure S. 6). The incidence of surgical sepsis was greatly reduced and this became a common practice. This also provided indirect evidence for germ theory of disease. His experiments were the origin of the present-day aseptic techniques used to prevent infections. Contributions of Robert Koch and Germ Theory of Disease In Germany, Robert Koch (1843-1910, Figure S. 7) confirmed Pasteur’s germ theory and took it several steps further.
His investigations began with a study of Bacillus anthracis, which causes a disease in cattle. He cultured the anthrax bacillus and later used the same techniques in tracking and culturing the organism responsible for tuberculosis and cholera. Koch won the Nobel Prize 9for his work on tuberculosis, but is perhaps better remembered for his formulation of four basic principles or postulates of bacteriology known as Koch’s postulates and they are: Figure S. 6 [A] Joseph Lister, [B] Lister’s carbolic spray in use during an operation Figure S. 7 Robert Koch (1843-1910) 101.
Microorganism must be present in every case of the disease. 2. Microorganism must be isolated from the diseased host and grown in pure culture. 3. The specific disease must be reproduced when a pure culture of microorganism is injected into healthy susceptible host. 4. Microorganism must be recovered once again from experimentally inoculated host. He then carried out a series of experiments to demonstrate biological specificity of disease causing agent. In the mean time Pasteur also undertook work on Anthrax and reached conclusions similar to findings by Koch. In 1880, Pasteur used Koch’s technique to isolate and culture the acteria that caused chicken cholera. To prove his discovery, he arranged a public demonstration of the experiment that had been repeated successfully in the laboratory. He injected healthy chickens with pure culture of cholera bacterium and waited for them to develop the symptoms and die. But the chickens survived. Pasteur, on reviewing very carefully each step soon, found an explanation. He discovered that bacteria, if allowed to grow old, could become avirulent. But these avirulent bacteria could still stimulate something in the host to resist subsequent infection and immunize the host to that disease.
He then applied this principle of immunization for prevention of anthrax, and again succeeded. He called these avirulent cultures as vaccines and the process of immunization with such cultures as ‘vaccination’. This recognized the earlier work of Edward Jenner, who had successfully vaccinated a boy named James Phipps against smallpox in 1798 (Figure S. 8). Jenner had observed that milkmaids exposed to cowpox never developed the serious smallpox, and thus hypothesized that exposure to cowpox somehow led to protection against smallpox.
In order to prove his point, he inoculated James Phipps first with cowpox material, and later with smallpox- causing material. The boy did not get smallpox. Figure S. 8: Edward Jenner vaccinating James Phipps with cowpox material Pasteur by now was considered as miracle worker in France and was asked to develop vaccine against rabies- a disease transmitted to humans from the saliva of infected dogs, cats and wolfs. Pasteur took the challenge and reproduced the disease in rabbits by inoculating them with saliva 11from rabid dogs.
He then removed its brain and spinal cord, dried and pulverized them, and mixed the powder into liquid and successfully vaccinated dogs with a series of shots of this mixture. In 1885, a boy named Joseph Miester, who was bitten by a rabid wolf, was inoculated by Pasteur with his vaccine, which saved his life (Figure S. 9). When he saved most of the group of Russian farmers bitten by rabid wolves, the Czar sent him 100,000 francs, which along with many other donations from around the world, founded the famous Pasteur Institute in Paris. Figure S. 9 Joseph Miester being examined by Pasteur’s associate, as Pasteur looks up
Rise of Medical Microbiology The work on Anthrax and rabies started the golden age of medical bacteriology. The Pasteur Institute in Paris and Institute in Berlin became the world centers of science of Bacteriology. Robert Koch developed methods for isolation of pure cultures of bacteria and concentrated his work more on isolation, cultivation and characterization of disease causing agents of major diseases in man. On the other hand, Pasteur focused his attention on seeking experimental evidence of how infectious diseases occur in human body and how recovery and immunity occurs.
This was a great medical revolution and within 25 years, methods for prevention by immunization or hygienic methods were developed against most of the major bacteria causing human diseases (Table 1). Discovery of Filterable virus At Pasteur Institute, filters to retain bacterial cells were developed and were used to get filtrates free from bacterial cells. In 1892, a Russian scientist Dmitri Iwanovski filtered infectious extract from tobacco plant infected with mosaic disease. To his surprise he found that filtrate was still fully infectious.
This specific discovery was confirmed soon and within few years, many other plant and animal diseases were found to be caused by some submicroscopic agents retained 12in the filtrates passed through bacterial filters. A new class of infectious agents was discovered and these were called ‘viruses’ (from latin word virus, meaning a slimy liquid or a poison). It was later found that these agents are different from cellular organisms already known in structure and development. Frederick Loeffler and P Frosch (1898) found that Foot and Mouth disease (FMDV) is caused by a virus.
Bacterial viruses were independently discovered later by F. W. Twort and F. d’ Herelle (1917). Stanley (1935) crystallized virus and found that it is made up of protein and nucleic acid. Table 1: Some Early Discovery of Bacteria Causing Human and Animal Diseases Date Disease Causative agent* Discoverer 1876 Anthrax Bacillus anthracis Koch 1880 Typhoid fever Salmonella typhi Eberth 1880 Malaria Plasmodium spp. Laveran 1882 Tuberculosis Mycobacterium tuberculosis Koch 1880 Cholera Vibrio cholerae Koch 1883 Diphtheria Corynebacterium diphtheriae Klebs and Loeffler 1885 Tetanus Clostridium tetani Nicolaier 887 Meningitis Neisseria meningitidis Weichselbaum 1894 Plague Yersenia pestis Kitasato and Yersin 1896 Botulism Clostridium botulinum Van Ermengem 1898 Dysentry Shigella dysenteriae Shiga 1905 Syphilis Treponema pallidum Schaudinn and Hauffman 1906 Whooping cough Bordetella pertussis Bordet and Gengou * Present name and original names of Causative agent, in many instances, was different. General characteristics of Virus 1. They are obligate parasites and can grow and multiply in living plant, animal or bacterial cell as host. Such viruses are known as plant, animal and bacterial virus respectively. . They are nucleoprotein particles made up of protein and nucleic acid. 3. They have only one type of nucleic acid either DNA or RNA. No virus contains both. 4. They are ultramicroscopic and can only be seen with the help of an electron microscope. 5. They do not have cellular structure and unit of structure is ‘virion’ Chemotherapy The methods to control and prevent various diseases became very important and the use of chemicals to kill or inhibit the disease causing organisms was termed chemotherapy. Chemotherapy has been practiced for hundreds of years.
Mercury was used toto treat syphilis as 13early as 1495, and cinchona bark was (Quinine) was used in South America since 17th century to treat malaria. Paul Ehrlich (1909, Figure S. 10) discovered a synthetic arsenic compound (606th compound) named Salvarsan that proved effective against syphilis bacterium. The discovery of antibiotic (penicillin) by Alexander Fleming (1929, Figure S. 11) opened another era for chemotherapy to control infectious diseases as they were found to be most effective chemotherapeutic agents against bacterial diseases. The discovery of penicillin was an accidental one.
One day Alexander Fleming noticed that bacteria were not growing near the mold in some of the mold contaminated culture plates. This was identified and established to be Penicillium notatum. He correctly guessed that the mold was producing some substance that was inhibiting bacterial growth. His original report went unnoticed for almost ten years, when a group from Oxford University, led by Howard. W. Florey and E. Chain, conducted clinical trials with penicillin to prove it as a ‘miracle drug’. Florey, Chain and Fleming later were awarded Noble Prize in 1945 for their work.
Another major discovery in chemotherapy occurred in 1932, when, G. Domagk, a German physician, discovered a group of chemicals called sulfonamides, or sulfa drugs, which were observed to be effective against various kinds of bacterial infections. Domagk won the Noble Prize for his discovery which helped to launch a second wave of research on chemotherapeutic agents. Now hundreds of new antibiotics have been discovered for the treatment of several infectious diseases. Development of vaccines since then has become a continued process and an industry by itself. Figure S. 0: Paul Ehrlich Figure S. 11: Alexender Fleming Development of pure culture methods Around 1870 it was realized that pure cultures must be used for proper understanding of form and function of microorganisms. 14What is a pure culture? A pure culture of an organism is the culture which contains large population of only one type of microorganism generally developed from a single cell. Brefeld introduced the practice of single cell isolation and cultivation of fungi on solid medium containing gelatin as solidifying agent. Joseph Lister developed serial dilution technique for pure culture isolation.
He devised a small syringe prototype of the modern micropipettes to dispense small aliquots of liquid in different tubes containing milk so that final dilution contained one or none of the organism and isolated pure culture of bacteria which was confirmed by microscopic examination. Robert Koch was experimenting with solid media and used sterile cut surfaces of potato placed on sterile covered plates to grow bacteria. Since the surface was opaque it was difficult to examine cultures of bacteria. Richard. J. Petri introduced Petri dish as a suitable medium container for the culture of bacteria.
Pour plate and streak methods for the isolation of pure cultures were also developed by Koch. The use of gelatin as solidifying agent had following disadvantages; viz. , It is a protein which is susceptible to microbial digestion and changes from gel to liquid at 280 C while the optimum temperature for growth of wide range of bacteria is between 30 – 370 C. Fran Hesse introduced agar- a complex polysaccharide extracted from red algae as the solidifying agent, which was found to be a suitable solidifying agent because of following properties; a) It is not digested by bacteria easily and its melting point is 1000
C and remains solidified below 44 0 C, besides producing a transparent stiff gel and growth of bacterial/fungal colonies can be seen easily on their surface. Microorganisms as Geochemical Agents The pioneering work by Sergei Winogradsky and M. W. Beijerinck (1851-1931) showed that microorganisms exhibit wide range of physiological diversity and carry out chemical transformations that can’t be performed at all by plants and animals. They play important role in cycles of C, N and S etc and are responsible for turnover of matter on earth.
They discovered a unique class of bacteria called chemoautotrophic bacteria which can grow in complete inorganic environments getting energy by oxidation of reduced inorganic compounds and carbon from carbon dioxide. Important bacteria in this category are sulfur bacteria which oxidize inorganic S compounds and nitrifying bacteria which oxidize inorganic N compounds. They also developed enrichment techniques to culture specific bacteria and their isolation. Nitrogen fixing Bacteria
Microorganisms play an important role in the fixation of atmospheric nitrogen which cannot be used directly as source of N2 by most living organisms. Symbiotic N2 fixing bacteria (Rhizobium) and asymbiotic N2 fixing bacteria (Azotobacter) which use gaseous N2 for synthesis of their cell constituents help to maintain supply of combined N2 on which all other forms of life are dependent. These kind of bacteria were also discovered by Winogradsky and Beijerinck. Microbiology in the Twentieth Century
In the 20th century, studies on microorganisms have contributed towards development of other disciplines such as industrial microbiology, biochemistry and molecular genetics etc. The discovery of cell free alcoholic fermentation by H. Buchner in 1897 laid down the foundation for the beginning of Biochemistry. The discovery that vitamins used by animals are similar to growth factors required by bacteria led to the finding that there is similarity of metabolism in all 15living systems and hence microorganisms were used as models to understand basic fundamental metabolic processes.
Escherichia coli has been extensively used in this category to understand biochemistry and genetics of various cellular processes. George Beadle and Edward Tatum (1941, Figure S. 12) obtained mutants of bread mold Neurospora and studied the consequences of permanent genetic changes in biochemical terms. Oswald Avery, Colin Macleod and Maclyn Mc Carty (1944) proved that DNA is the basic genetic material and a model was proposed for the molecular structure of DNA by James Watson and Francis Crick (1953, Figure S. 13).
Collapse of the boundaries between the subjects such as Microbiology, Genetics, and Biochemistry has lead to deeper understanding of biology at molecular levels under new discipline called Molecular Biology and Genetic Engineering. The application of Molecular biology has revolutionized the use of genetically engineered microorganisms for technological purposes. Now microorganisms are being used to produce non-microbial products at commercial scale for the welfare of human beings. Production of injectable insulin by genetically engineered E. oli has opened the possibilities for search and development of other suitable organisms for production of useful products on industrial scale. The exploitation of microorganisms, their systems or their processes for technological purposes is studied under Microbial biotechnology or industrial microbiology. Figure S. 12: George Beadle and Edward Tatum Activities of microorganisms important in our daily life Microorganisms play an important role in sustaining life on this planet and in our daily life through the following activities: . Transformation of matter: Microorganisms degrade dead organic matter and return to the atmosphere in inorganic form. They complete the cycle of matter and are responsible for transformation of C, N and S and other important elements which are essential for life. 16Figure S. 13: Watson and Crick 2. Biological nitrogen fixation: They fix nitrogen from atmosphere and make it available to the plants in usable form. Important microorganisms under this category include, Rhizobium, Azotobacter, Azospirillum etc. 3.
Mycorrhiza: Association of roots of many plants with fungi forms a composite structure called mycorrhiza. Fungus helps in absorption of mineral salts from soil and plant in turn provides carbohydrates for the growth of fungus. 4. Silage: This method is used to preserve feed with its characteristic flavor, taste and nutritive value. Leaves of green plants are compacted in size and some molasses is added. Lactic acid bacteria develop and produce lactic acid which helps to conserve the cattle feed. 5. Cellulose degradation in Rumen: Ruminants feed on straw and grass which contains about 50 % cellulose.
There is symbiotic association of microorganisms with rumen for degradation of cellulose and about 1010 – 10 11 cells/ml of different bacteria are usually present in the rumen. Most important of these include Ruminococcus andClostridium. 6. Biogas: Animal waste products and cellulose containing waste is fermented by microorganisms (Methanogens). N2 of the animal excreta is preserved in rotting sediment and methane gas so formed is used as a fuel. 7. Composting: Decomposition of organic matter by microorganisms to convert it into nutrient rich manure is known as composting.
Bacillus, Aspergillus and Thermoactinomyces are important in this process. 8. Industrial uses: Different microorganisms are used for the production of wide range of products at industrial scale. These include alcoholic beverages, antibiotics, enzymes, pharmaceuticals etc. Scope of Microbiology Depending on their applications in different fields, the major areas of applied microbiology are: 17Agricultural Microbiology/ soil Microbiology: Microorganisms related to soil fertility, plant diseases, transformation of matter, biological nitrogen fixations etc are studied.
Food Microbiology: Microorganisms important with respect to food viz. , food fermentations, food spoilage, food poisoning and food preservation are studied in this area. Industrial Microbiology: Microbial production of useful products like antibiotics, fermented beverages, alcohols, industrial chemicals, organic acids, enzymes, hormones etc are studied in this area. Medical Microbiology: Besides their usefulness, microorganisms are casual agents of several diseases of plants, animals and human beings. Many diseases are caused by viruses also.
Medical Microbiology deals with studies on causative agents of disease, diagnostic procedures, identification of disease causing organisms, development of effective vaccines and preventive measures etc. Exo-Microbiology: It deals with exploration of existence of biomolecules and microbial life in outer space. Geochemical Microbiology: prospects for deposits of coal, mineral and gas, recovery of minerals from low grade ores, sea water mining operations, coal, mineral and gas formation and exploration are studied in this area.
Molecular Biology: is the program of interpreting the specific structure and function of organisms in terms of their molecular structures. Microorganisms have been used as a tool to explore fundamental life processes because of many advantages; their fast rate of reproduction, their growth can be easily manipulated, and lysed cells can be studied in terms of specific chemical reactions, specific products and specific structures involved.
Genetic engineering and Biotechnology: This is an important development in applied Molecular Biology which refers to the human capability to alter the genetic make up of an organism. It has been possible because of the detailed knowledge of structure and function of DNA and discovery of the restriction enzymes which can cleave or cut the DNA at specific sites along the chain length. Use of genetically engineered microorganisms has opened great potential for production of drugs, vaccines, improvement of agricultural crops etc.
Environmental Microbiology: deals with use of microorganisms to protect the environment from the toxic pollutants, reduction of microbial load in the sewage and industrial wastes, pesticides, insecticides, heavy metals etc. and to develop suitable methods for treatment of these wastes and their recycled use. Some recent applications of bacteria Biosensors: Bacteria and their components are being used to detect toxic pollutants and continuous monitoring of nutrients/other parameters during food processing and fermentation processes.
Technically a Biosensor is a miniaturized analytical tool comprising of highly specific biological sensing element i. e. either integrated within or associated with transducer which 18convert physiochemical interaction into a discrete or continuous digital electronic signals, which are proportional to single or related groups of analytes. The detection of toxic pollutants in soil, water and environment is important for the protection of human and animals. Biosensors using components from bacteria have been constructed which can detect biologically active toxic pollutants.
Such bacterial sensor requires both a receptor which is activated in the presence of pollutant and a reporter which will make this change apparent. Biosensors use lux operon from bacteria Vibrio or Photobacterium. This operon contains inducer and structural gene for enzyme luciferase. In the presence of coenzyme FMNH2, luciferase reacts with molecule to form enzyme substrate complex which emits blue green light changing FMNH2 to FMN. Hence a bacterium containing lux gene will emit light when the receptor is activated. This operon has been transferred to bacteria like:
Xanthomonas…to monitor progression of infection in plants. Bradyrhizobium… to monitor root nodule formation and development. Lactococcus…to detect the presence of antibiotics in milk. Photo bacterium… to detect toxic pollutants. E. coli……………to detect the presence of mercury in soil samples. Biosensors only help to detect the presence of pollutants and separate processes are required to remove these pollutants. They also find application in medical or diagnostics field or in food and fermentation operations for measurement of specific parameters.
In food industry, biosensors can be incorporated into food packages to monitor temperature abuse, loss of shelf life, microbial contamination, and to provide visual indicator to consumers of the state of the product at the time of purchase. Bioremediation: The concept of using microorganisms to remove pollutants is known as bioremediation. We can use either indigenous microorganisms or genetically engineered microorganisms. Pseudomonas and Bacillus are two most important and most commonly used bacteria for bioremedical purposes.
These bacteria can either turn pollutants into energy source that they consume or alternately they produce enzymes that break down these pollutants into less harmful molecular products. Studies are being carried out to develop efficient microbial inoculants for bioremediation of effluents from distillery, tanneries, textile and food processing industries etc. Greenhouse Gas bioremediation: There is great concern about the greenhouse effect. Water, methane and carbon dioxide in earth atmosphere are important as they absorb infra-red radiations. This process is important for maintaining the temperature of the earth’s surface.
In the recent years because of increased burning of the fossil fuels, carbon dioxide content in the atmosphere has increased many folds and has caused rise in the temperature on the earth as a result of the greenhouse effect. Global warming leading to melting of the icy polar caps, rise in sea level and flooding of major coastal areas, besides warmer air is causing chronic draughts in major crop growing areas as a result of modified global air circulation pattern. Some scientists believe that nature automatically takes care of the problem and they emphasize that extra carbon dioxide stimulates plant growth.
They also believe that increase in crop productivity after the industrial revolution may infact be due to the presence of CO2 in abundance in the atmosphere and may not be due to the use of fertilizers and pesticides. This 19increased plant growth may be the cause of increased methane in the atmosphere as methane is five times more effective as a greenhouse effect gas as compared to CO2. Concentration of methane has increased alarmingly in the atmosphere and has infact doubled in 100 years which may be due to more plant growth resulting in more decaying plant material.
When dead plants decay under anaerobic conditions bacteria break down organic matter and release CO2 and H2 which is combined to form CH4 and H2 O by the activities of anaerobic methanogenic bacteria. The use of aerobic methanotrophic bacteria which oxidize methane is important and is being studied for greenhouse gas bioremediation. The activities of methanogens and methanotrophic bacteria should be balanced under ideal conditions and the use of chemical fertilizers and pesticides may have inhibited the growth of methanotrophs.
There is a need to determine the optimum growth conditions for these bacteria for use in greenhouse gas bioremediation. Suggested Reading 1. Pelczar, M. J, Chan, E. C. S & N. R. Krieg. Microbiology- Concepts and Applications (International Edition), McGraw- Hill Inc. 2. Stanier, R. Y. , Ingraham, J. L. , Wheelis, M. L. & P. R. Painter. General Microbiology. Fifth Edition, MacMillan. 3. Prescott, L. M, Harley, J. P & D. A. Klein. Microbiology. Third Edition, WCB Publishers. 20