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.

4. Fermentation (A biological Process); Pasteurization (Heating Destroys Microbes): Fermentation is a process that breaks down carbohydrates into alcohols and organic acids. Earlier it was believed that the fermentation is purely a chemical process.

But, it was Louis Pasteur, a chemist by training who convinced in 1857 the scientific world of his time that all fermentative processes are the results of microbial activity. This he did by showing that fermentation is invariably accompanied by the development of microorganisms.

Pasteur studied various types of fermentations and demonstrated that each particular type of fermentation occurs by the act of specific type of microorganism.

During his work on butyric fermentation Pasteur discovered another fundamental biological phenomenon: the existence of forms of life that can live only the absence of free oxygen (anaerobic life) and he introduced the terms ‘aerobic’ and ‘anaerobic’ to designate, respectively, life in the presence of and in the absence of oxygen.

It was found that during fermentation processes for desired products certain undesired microbes grew in “ferments” and resulted in undesired products. For convenience, rod-shaped bacteria grew in certain wine- vats and produced lactic acid that caused souring of wine.

To solve this problem, Pasteur (1860) suited that heating could be used to kill undesirable type of microbes growing in “ferments”. Pasteur’s this suggestion later came to be recognized as pasteurization, i.e., heating at moderate temperature to kill high percentage of microbial population. Today pasteurization is widely used in the fermentation industries especially the dairy industry.

5. Germ Theory of Disease: Though strong arguments for the germ theory of disease (diseases are caused by living organisms) were given by many earlier workers but all were mostly speculative. The first satisfactory demonstration of the probable causal relationship of organisms to disease was given by Benedict Prevost (1807) in plants He proved conclusively that bunt disease of wheat is caused by a fungus.

Though Prevost’s experiments provided the first proof and interpretation of the role of a microorganism in the causation of a disease (i.e. the germ theory of disease), his findings were ahead of his time and were rejected by almost all his contemporaries who believed in spontaneous generation of microorganisms and of disease and that the microorganisms and their spores were the result rather than the cause of disease.

The discovery that bacteria can act as specific agents of infectious disease in animals was made through the study of anthrax disease. C.J. Davaine (1863 and 1868) showed that bacteria are invariably present. In diseased animals but are undetectable in healthy ones and that the disease can be transmitted to healthy animals by inoculation with blood containing these bacteria but he could not prove whether these bacteria were the cause or the result of the disease.

Somewhat later, Robert Koch (1876) solved the problem and confirmed the germ theory of disease by conclusive demonstration of the bacterial causation or etiology of anthrax disease.

By a series of experiments on anthrax disease of cattle, he showed that the spores of anthraxbacilli isolated from pure cultures could infect animals and thus demonstrated that germ grown outside a body could cause disease and that specific microorganisms caused specific diseases.

Finally in 1882, to establish cause and effect relationship between a given microorganism and a specific disease, Koch described some steps necessary to identify the causative agent of a disease.

These steps are popularly known as Koch’s Postulates which is; even today used in animal and plant pathology.

In fact the “steps” were the ideas of Jacob Henle who proposed during 1840s that to establish the etiology of a specific disease, the agent would have to be found regularly in the host during the disease, the agent would have to be isolated, and the isolated agent would have to be shown capable of producing the disease. Koch, however, first applied these ideas experimentally and established their validity.

6. Pure Culture Concept and Other Microbial Techniques: Around 1870 it began to be realized that a sound understanding of the form and function of microorganisms could be obtained only if they are isolated and grown in pure culture form. A pure culture is one that contains only a single kind of microbial population grown from a single cell.

Much of the pioneer work on pure culture technique was done by B. Brefeld (a mycologist) but his methods worked well for fungi and were found to be unsuitable for bacteria.

It was Joseph Lister (1878) who first obtained pure culture of bacteria using serial dilutions in liquid media. He took a fluid containing a mixture of bacteria, diluted it with sterile medium, and delivered it into a container of sterile milk by a specially constructed syringe. After incubation, he found that there were single kind of bacteria growing in container identical to their parent cell. In practice, serial dilution method proved to be tedious, difficult and uncertain for routine use. It also proved to be disadvantageous because one could only isolate in pure form the microbes that predominated in the original mixture. Therefore, another promising device needed to be investigated.

Robert Koch was particularly concerned with this problem and, at first, he cultured bacteria on solid fruits and vegetables such as slices of boiled potato but many bacteria did not grow on such substrates. Then he perceived that it would be far better if a well-tried liquid medium could be solidified with some clear substance.

Koch (1881) tried gelatin as a solidifying agent and succeeded in developing solid culture media, but gelatin, the first solidifying agent used, had serious disadvantage of becoming liquid above 28-30°C which is below the optimum temperature for the growth of human disease producing bacteria.

However, Koch replaced gelatin by agar in 1883-84 on the recommendation of F.E. Hesse, a German housewife, who had gained experience with the characteristics of agar in the process of making jelly.

Agar is still frequently used as solidifying agent in microbiological laboratories. The development of solid culture media to grow pure culture was of fundamental importance and may be considered one of the Koch’s greatest contributions.

Besides developing solid culture media using gelatin and agar, Koch also evolved methods to place microbes on glass slides and colour them with analine dyes (stains) so that the individual cells could be seen more clearly under the microscope.

7. Growth of Medical Microbiology: 1. Causation of Diseases: Reports regarding causative agents of animal and human diseases started pouring in during the second quarter of the nineteenth century. A. Bassi (1836) recognised that the disease of silkworms may be caused by a fungus. J. Schoelein (1839) established that ‘favus’ is caused by a pathogenic fungus. D. Gruby (1843) revealed the causative agent of trichophytosis (ringworm).

In the second half of the nineteenth century due to availability of better microscopes and abandonment of spontaneous generation, Pasteur and his contemporary workers turned towards this area of microbiology which successfully led to several important discoveries regarding causative agents of animal and human diseases with certainty (Table 1.3).

2. Immunization: Immunization is the artificial induction of immunity to disease. The practice of immunization was used in Asia for centuries to produce immunity to smallpox before it was introduced into England in 1718 by Lady Mary Montagu but she had no explanation of how or why it worked.

The procedure of immunization against smallpox was quite simple; material from a pustule of an infected person was scratched into the skin of the person to be immunized.

In most cases this resulted in a mild case of smallpox without the scarring that was common in naturally acquired cases. E. Jenner (1749-1823) in 1798 reported to Royal Society in London the value of immunization with cowpox as a means of protecting against smallpox; a clear case of vaccination.

This he did on the basis of the fact that when he inoculated a 8- years old boy, James Phipps, with cowpox virus content, the boy escaped from small pox infection.

Jenner’s explanation regarding cowpox vaccination against small pox established the scientific credibility of vaccination to prevent disease and was accepted by the scientists and physicians of the time.

Jenner, therefore, is credited to have develop first vaccine from cowpox (Latin name Vaccinia). Later on, Pasteur developed vaccines (attenuated microorganisms) against chicken cholera and anthrax in 1880, and against rabies in 1885.

3. Surgical Antisepsis: Joseph Lister (1827-1912), a British surgeon, reasoned that the post-surgery infections might well result due to microorganisms present in air that enter the tissues exposed during operation, and revolutionized surgical practice in 1867 by introducing antiseptic principles. He used carbolic acid (a phenol) as a disinfectant and adopted several other antiseptic procedures which are in practice still today.

4. Rise of Chemotherapy: For many years even after the discovery of the role of microorganisms as the causative agents of infectious diseases, their control was largely preventive, exclusively based on the use of vaccines and antisera and there were no scientific approach to cure them after they had appeared in an organism.

Paul Ehrlich (1854-1915), a German physician-chemist, undertook extensive studies to search synthetic chemicals having curative properties for pathogenic microorganisms and coined the term ‘chemotherapy’ to describe this approach to control infectious diseases.

During his research between 1880-1910 he developed almost 1000 new derivatives of an arsenical chemical, the atoxyl, and found that the derivative no. 606 called salvarsan proved to be effective in treating syphilis. In this way, Paul Ehrlich, the founder of modern chemotherapy, opened a new way of chemical treatment of infectious diseases.

8. Growth of Plant Pathology: It has been shown during the early decades of nineteenth century that specific fungi can cause diseases of wheat and rye, and in 1845 A.J. Berkeley stated that the blight disease of potato in Northern Europe during 1840s was caused by a fungus.

The blight disease of potato turned to epiphytotic form in the mid-1840s particularly in Ireland resulting in death and migration of great number of people and tragically dramatized the importance of plant diseases.

Extensive studies started and, finally, de Bary (1861, 1863) proved experimentally that the causal agent of blight of potato was a fungus which he named Phytophthora infestans de Bery thus pioneered the scientific approach in the area of plant diseases and is rightly called the “Founder of Experimental Plant Pathology”.

Burill (1878) for the first time showed that the fire blight disease of pears was caused by a bacterium, Erwinia amylovora. Infect, it was the discovery of phytopathogenic nature of bacteria. This achievement of Burill was established beyond any doubt with numerous and excellent contributions on the study of bacterial disease of plants by E.F. Smith from 1895 onwards.

9. Discovery of Viruses: The tobacco crop in Holland was struck by a severe disease around 1870. Adolf Mayer, Director of Agricultural Experimental Station, Wageningen, began his studies on this disease about 1880 and published his results in 1886.

Mayer christened the disease as “MOSAIKKRANKHEIT” (mosaic-like), from the mosaic-like pattern on leaves of diseased plants and succeeded in reproducing the disease by infecting juice extracted from infected tobacco leaves onto healthy ones; he could not succeed in identifying the real agent that caused the disease.

However, Mayer’s contribution will always be remembered as he was the first person who put first step forward in the development of a new discipline later recognised as ‘Virology’. A later in the year 1892, D. Ivanowski first successfully experimentally demonstrated that the tobacco mosaic disease has been caused by agents which successfully passed the Chamberland-filter that retains even the smallest bacteria.

It was an important clue, but contrary to his experimental result and despite his inability to isolate any bacterium. Ivanowski still maintained that either the ‘pathogenic bacterium’ somehow passed through the filter or a ‘toxin’ secreted by them passed through the filter and made the filtrate infectious. Within six years after the experiments of Ivanowski.

M. Beijerinck (1898) confirmed by repeating the same experiments and found that the tobacco mosaic disease was caused not by any pathogenic bacteria or toxin but rather by some new type of pathogenic agents which he called “contagium vivum fluidum” (infectious living fluid) and referred subsequently to it as a “virus” (poison). He also said that the viruses multiply only inside the living cell.

10. Other Important Contributions: The methyl violet dye was first used by Weigert (1875) for staining bacteria. Christian Gram (1884) introduced the ‘Gram Staining Technique’ to stain bacteria.

We still use this technique for identifying and classifying bacteria. H.C. Hansen (1842-1909) opened the way to study of industrial fermentations as he developed the pure culture study of yeast and bacteria used in vinegar manufacture and named them as “starters”.

Adametz (1889), for example, used pure cultures in cheese manufacture, and Conn and Weigmann developed pure culture starters for butter production (1890-1897).

Thus the science of microbiology grew up profoundly within a period of forty years (1860-1900). Its early years were tempestuous, but by the first years of twentieth century the man in the street and on the farm knew of bacteria. Man, by this time, also knew that the bacteria could do good for him, and resolved in learning to control them from causing diseases to plants, animals, and humans.

Many landmark discoveries in the field of microbiology have been made in 20th century, the modern period of grow of microbiology. However, the landmark events in the development of microbiology are given in Table 1.4.

Branches of Microbiology: 1. Industrial Microbiology: It encompasses the uses of a variety of microbes in industrial processes. Initially they were being used for industrial fermentation and waste water treatment. As today industry is linked to biotechnology, several new industrial applications have been found for a variety of microbes.

It is sometimes also studied as microbial biotechnology and is the application of scientific and engineering principles to the processing of materials by microorganisms (such as bacteria, fungi, algae, protozoa and viruses) or plant and animal cells to create useful products or services. Areas of industrial microbiology include quality assurance for the food, pharmaceutical, and chemical industries.

2. Medical Microbiology: This branch of microbiology deals with the scientific study of pathogenic microbes, the diseases they cause, their mode of survival in environment and their hosts (including life-cycle); their diagnosis, prevention and treatment.

In fact, as early as Varo and Columella in the first century BC had postulated that the diseases were caused by invisible beings (animalia minuta). Von Plenciz (1762) had put forth the idea that each disease was caused by a separate agent. It covers a variety of topics where microbes are responsible for causing diseases of skin and eye infections, pneumonia (by bacteria), several sexually transmitted diseases (STD’s), minor arthropod diseases, gastrointestinal infections including infections from drinking cow milk harbouing certain pathogenic bacteria and their remedies etc.

3. Agricultural Microbiology: This branch deals with microbes having an impact on agriculture and food chains. Both, the harmful

(microbes causing plant diseases) as well as useful microbes (e.g., N2 fixing microbes, use of microbes in bio-fertilizers etc.) are studied under this branch. Certain raminants also carry a mixture of complex bacteria that enable the animal to extract sufficient nutrient from a diet of grasses. Future research in microbial ecology will help to determine in preserving a balance in mirobial communities that favour agriculture.

4. Environmental Microbiology: In the late 1800’s, and early 19th century Sergei Winogradsky, a Russian Mineralogist, pioneered the field of microbial autotrophs, and initiated the field of Environmental Microbiology. This branch includes the study of composition and physiology of microbial communities of the environment.

It also deals with the activities of microbial entities, their interactions among themselves and with maroorganisms. Adhesion, biofilm formation, global element cycles, biogeochemical processes and microbial life in extremes of environment or unexplored environs all fall in its preview.

5. Food and Dairy Microbiology: As the microorganisms are ubiquitous (present almost everywhere) food and milk are no exceptions. Hence the microbes are studied from the viewpoint that they (e.g., Bacteria, Yeasts, molds etc.) can either act as spoilage microorganisms or pathogenic microorganisms.

And thus how they can cause spoilage, prevent spoilage through fermentation or can be the cause of human illness, all comes under the realm of this branch of microbiology. It is a thrust area of microbiology these days, as more and more food items are being packaged (including milk and its products) for later use.

6. Biotechnology: The UN convention on Biological Diversity defined Biotechnology as: any technological application that uses biological systems, living organisms, or derivatives thereof to make or modify products or processes for specific use. Bio-engineering, including recombinant genetic technology of the 21st century is the science upon which all biotechnological applications are based. It combines disciplines like genetics, molecular biology, biochemistry, food sciences, mechanical engineering, chemical engineering, microbiology, cell biology and all are interrelated to electronics, information technology and robotics.

7. Bacteriology: The current science of bacteriology includes the study of both domains of prokaryotic cells (the Bacteria, and Eucarya). But recently due to out-break of molecular techniques applied to phylogeny of life, another group of prokaryotes was defined and informally named,” archaebacteria (it has now been renamed as Archaea) and included in the study of bacteriology”. 8. Virology: It is the study of viruses, complexes of nucleic acids and proteins that have the capacity for replication in animal, plant or bacterial cells. To replicate, the viruses use their genomes (DNA or RNA) or of the host cells and cause changes in cells, particularly its antigenicity and may cause several diseases in plants and animals is all covered under this branch.

9. Soil Microbiology: This branch deals with the biota that inhabits the soil and the processes they mediate. As the soil is a complex environment, colonized by an immense variety of microorganisms, the soil microbiology focuses on soil viruses, bacteria, actinomycetes, fungi and protozoa, but traditionally it has also included investigations of soil animals such as nematodes, mites and other arthropods.

Modem soil microbiology represents an integration of microbiology with the concepts of soil science, chemistry and ecology to understand the functions of microorganism in the soil environment.

10. Sewage Microbiology: This branch deals with the study of microbial flora of various types of sewage. The sewage may, depending upon source, can contain harmless (E. coli and other coli forms) to potential pathogens including enterococci, Vibrio cholerae, Salmonella typhi, etc. This branch studies their qualitative as well as quantitative details and ways to combat them following various treatment processes.

11. Mycology: It deals with the study of various fungi. Fungi are eukaryotic organisms and around 300 species are shown to be pathogenic for man. It studies their morphology, taxonomy, biosystematics, distribution, propagation, and several mycotic diseases they cause including hypersensitivity, mycotoxicoses, mycetismus and other infections and their remedies.

12. Phycology: It is a sub-discipline of botany, and deals with the scientific study of algae. As many species are single celled and microscopic (e.g. Phytoplankton and micro algae); yet others are multicellular (some growing) very large as seaweeds such as Kelp and Sargassum they are also studied in microbiology.

It also covers aspects like cyanobacteria (blue-green algae) and other microscopic forms occurring as symbionts in lichens.

13. Protozoology: Earlier much in use, this branch is the study of protozoa (motile and heterotrophic) protists. Protozoa- despite their small size and unicellularness offer complex and unique biological features. They also serve as experimental models in a variety of cellular, molecular, biochemical and ecological researches.

One of the applied sub-branches of this old branch is medical protozoology (covering protozoa infecting humans). It covers life-cycles, morphological features, host-parasite interactions, geographical distributions, reservoir hosts, method of transmission and control, pathology, immunological aspects, diagnosis and remedies are all included in it.

14. Aquatic Microbiology: It covers the study of microorganisms and their activities in natural water. As the natural waters include lakes, ponds, streams, rivers, estuaries and oceans, it initially started covering all of them.

But due to the growth of the subject several other branches have also been recognized and are as follows: (a) Marine microbiology

(b) Estuarine microbiology

(c) Groundwater microbiology, and

(d) Deep-sub-surface microbiology

The aquatic microbiology deals with the variability of aquatic habitat and rapid changes in characteristics and associated microbial component. The microbial activity and biomass measurement studies are performed to follow microbial functions in water ecosystems. Also the balance between N, P, O and H is studied in lakes, eutrophic systems, and streams etc.

15. Marine Microbiology: As marine environment is the largest part of the biosphere, being about 97-98% of all the water on earth, efforts are being made to study seas, especially deep seas (and their microbial functioning), as 75% of the ocean is below 1000 m depth (and is constantly cold at about 3°C on an average).

The oceanic explorations like Challenger expedition and Galathea expedition were among the initial efforts to critically explore microbial aspects of the deep seas and the nature of psychrophilic bacteria. Barotolerant bacteria are among the unique fauna of deep oceans.

As the most of the earlier work on seas and oceans remains confined to the near-shore and estuarine marine environments, the interest is growing in the off-shore and pelagic ocean microbiology.

Applications of Microbiology Some of the major applications of microbiology are as follows: Microbiology is one of the most applied branches of science. Its outstanding applications in the field of food microbiology, medical microbiology, industrial microbiology, soil microbiology, water and wastewater microbiology, microbial technology (biotechnology), extraction of metals and environmental microbiology including the use of microorganisms as biosensors is as given below.

1. It provides us with information about different types of microorganisms enabling us to understand their structure and functions; identifications and differentiations; their classifications; nomenclatures (naming), requirements regarding their nutrition; their isolation and purification; as plant and human pathogens; to derive phylogenetic relationships (relationships according to developmental stages in the evolution of an organism) and to understand the origin of life itself.

2. Microorganisms as food: Besides comestible fungi like mushrooms, microorganisms are also being used as single cell protein in the form of yeasts, bacteria, cyanobacteria, fungi as human food or animal feed. The production of the algal microbes as Chlorella (green alga and Spirulina (cyanobacterium) are being produced in Japan, Taiwan, Mexico, Israel, Thailand and America. Production of cellulose or lignocellulose utilizing microorganisms serves as human food as such or in the form of their products. Microbial products are also used as animal feed.

3. Microorganisms are used in production of a large number of, fermented foods such as leavened bread, sourdough bread, fermented milk products and flavours. The fermented milk products are yoghurt, cheese and several other products.

4. The important fermented vegetables are sauerkraut (from cabbage) and Kimchi (from other fermented vegetables in Korea).

5. Fermented meats and fermented fish are used in different parts of the world due to their increased retentivity, otherwise the meats and fish are highly perishable.

6. Beer, vinegar, tempeh, soya sauce, rice wine too are fermented products.

7. Microbiology has been very useful in preservation of food by heat processing, by pasteurization and appertization (commercially sterile food), by calculating thermal death values, prevention of spoilage of canned foods, aspectic packaging, irradiation, UV radiation, ionizing radiation, high pressure processing, i.e., pascalization, low temperature storage (chill storage and freezing), chemical preservatives (organic acids, esters, nitrite, and sulphur dioxide).

In food microbiology one learns about bacterial and nonbacterial agents of food borne illness. Among the helminthes and nematodes are: Platyhelminthus (i.e. liver flukes and tapeworms) and roundworms (e.g., Trichinella spiralis). The protozoa that cause food borne diseases are Giardia lamblia and Entamoeba histolytica.

8. Microbial diseases: Microorganisms are the causative agents of a large number of diseases which have been described under a separate chapter.

9. Industrial Microbiology: A large number of products of microbial metabolism after microbial processing of raw materials are produced on industrial scale. A separate chapter has been given on ‘Industrial Microbiology’.

10. Energy from microbial sources: A number of substrates can be used as a source of energy as biogas from methanogenic microorganisms. The microbes like Methanobacterium and Methanococcus can utilize CO2 as an electron acceptor finally producing methane. A new species of Methanobacterium, i.e., M. cadomensis strain 23 has been evolved in Japan for faster production of methane. Ethanol can also be used for the production of gasohol by mixing 80 per cent gasoline and 20 per cent ethanol. 11 .Degradation of cellulose and lignin:

Trichoderina reesei can be used to degrade cellulose since it produces extracellular cellulase. The white rot fungus Sporotrichum pulverulentum is a cellulase negative organism but a mutant of it has been prepared which can degrade kraft and wood lignocellulase actively. It has been possible to produce biological pulp without any chemical treatment for delignification.

12. Mining and extraction of metals:

Thiobacillus ferrooxidans and combination of Leptospirillum ferroxidans and Thiobacillus organoparpus can be used to degrade pyrite (FeS2) and chalcopyrite (CuFeS2). The archaeal species Sulfolobus acidocaldarius and S. brierlevi are capable of oxidizing sulphur and iron for energy depending on C02 or other simple organic compounds for carbon. The pyrite and chalcopyrite are also degraded by these archaeobacterial species. 13. Recombinant DNA and genetic recombination:

Recombinant DNA is a wonderful product of genetic engineering, i.e., manufacturing and manipulating genetic material in vitro. The process of joining DNA from different sources is genetic recombination. A large number of restriction enzymes/restriction endonucleases have been obtained from various microorganisms that can cut or cleave double stranded DNA leaving staggered ends.

14. Hybridoma and preparation of monoclonal antibodies:

Hybridoma is a cell made by fusing an antibody-producing B-cell with a cancer cell. The resulting hybrid myeloma or hybridoma cells have properties of both parent cells immortality and the ability to secrete large amounts of a single specific type of antibody. This was discovered by Kohler.

15. Harvesting DNA biotechnology for public health engineering programmes:

Such programmes include production of interferon which is an antiviral protein produced by certain animal cells in response to a viral infection, production of human insulin production of somatotropin a human growth hormone and production of a large number of other hormones and vaccines.

The vaccines for cholera, diphtheria, tetanus, pertussis, viral hepatitis type A, type B, influenza, mumps, measles (rubella) plague, poliomyelitis, rabies, rubbela, typhoid, typhus and yellow fever have been developed so far.

16. Microbial technology of nitrogen fixation exploiting symbiotic microorganisms in association with lower or higher plants and asymbiotic or nonsymbiotic (by nitrogen fixing microorganisms independently).

Detailed information is covered under a separate chapter on ‘biofertilizers’. In nature, in legume root nodules a red pigment containing protein called leghaemoglobin is involved in the process of nitrogen fixation. The key enzyme responsible for biological conversion of molecular nitrogen to ammonia is nitrogenase. 17. Making faster and smarter computers:

The Archaeobacterium Halobacterium halobium grows in nature in solar evaporation ponds having high concentration of salts. Such salty ponds are found around San Francisco Bay located on the Western coast of USA.

It has been found that the plasma membrane of Halobacterium halobium fragments into two fractions, when the cell is broken down. These two fractions are red and purple. The purple fraction is important in making computer parts (chips). The purple colour is due to a protein which is 75% of purple membrane and has been referred to as bacteriorhodopsin.

Robert Birge at Syracuse University’s Centre of Molecular Electronics has grown Halobacerium halobium in 5-litre batches and has extracted the protein bacteriorhodopsin from the cells and developed the computer chips which are made up of a thin layer of bacteriorhodopsin.

The chips so made from the bacterial source can store more information than the conventional silicon chips and process the information faster more like a human brain. The only drawback is that one needs to store the protein chips at -4°C. But Birge believes that this problem will be overcome soon.

Techniques of Sterilisation Sterilisation by Heat: Heat is the most widely used lethal agent for sterilisation. Objects may be sterilised by dry heat, applied in an oven in an atmosphere of air or by moist heat, provided by wet steam.

Of the two methods, sterilisation by dry heat requires a much greater duration and intensity. Heat conduction is less rapid in air than in steam. Dry heat is used principally to sterilize glassware or other heat stable solid materials. But steam must be used for heat sterilisation of aqueous solutions.

Autoclave: Steam sterilisation is usually carried out in a metal vessel known as autoclave, which can be filled with steam at a pressure greater than atmospheric pressure. Sterilisation can thus be achieved at temperatures considerably above the boiling point of water; laboratory autoclaves are commonly operated at steam pressure of 15 lb/in2, above atmospheric pressure, which corresponds to a temperature of 120°C.

The autoclave is a cylindrical metal vessel having double walls around all parts except the front to withstand the high steam pressure. The base of the inner cylinder is concave, which contains water. The vessel is heated by immersion heater.

There is a strong lid covering the cylinder which can be tightly screwed. The lid is provided with pressure gauze, safety valve, a steam-outlet and metal handles. Inside the vessel cavity, there is a wire basket with triple stand where the materials are packed before the start of sterilisation (Fig. 1.1).

At the start of the operation, all the air present in the chamber must be expelled and replaced by steam; this is achieved by the use of steam trap, which remains open as long as air passes through it but closes when the atmosphere consists of only steam.

If some air remains in the sterilisation chamber, the partial pressure of steam will be lower than indicated on the pressure gauge, and the temperature will be correspondingly lower. For this reason an autoclave should always be equipped with both a temperature & pressure gauge. After adjustment of pressure (151bs/in2) inside the autoclave, the pressure is maintained for 15 minutes and then the autoclave is cooled.

Hot Air Oven: It is an electrically operated oven used for sterilisation of glassware viz. petridishes, flasks, tubes, pipettes etc. The apparatus consists of a large, rectangular, copper-base and covered with asbestos sheets. It is also provided with a door and erected on a four-legged stand.

The roof is provided with a hole through which a thermometer is fitted inside for recording of temperature. The oven has two or three shelves. The oven is heated by electrically operated heater, fitted at the base of the instrument. There is a regulator of heater to control the inside temperature (Fig. 1.2).

Before sterilisation, the glassware are dried properly and wrapped in brown paper and then exposed to hot air inside the oven. After loading of glassware, the oven is switched on 160°C.

The temperature will increase slowly up to the desired point where it will remain steady. Then at 160°C the oven is kept for an hour. This is the appropriate temperature for sterilisation of glassware. Then gradually the temperature is brought down and thereafter sterilisation is complete.

Red Hot Heat: This procedure is generally followed during working inside the culture room with inoculating needle. The inoculating needle is flamed to make it red hot. At that high temperature of the flame, all the organisms present on the needle are killed.

Technique # 2. Sterilisation by Chemical Treatment: Many of the substances used in preparing culture media are too heat liable to be sterilised by autoclaving. For such substances, a reliable method of chemical sterilisation is extremely useful.

The essential requirement for a chemical sterilisation agent is that it should be volatile as well as toxic, so that it can be readily eliminated from the object sterilised after treatment. Alcohol, Dettol, Lysol, ethylene oxide and various other volatile chemicals are used for this purpose.

Technique # 3. Sterilisation by Radiation: In recent years, various kinds of ionizing and non-ionizing radiation techniques have been employed for sterilisation of culture room, equipment’s etc. Exposure to UV radiation for a period of 45 min., is made. UV radiation is made by the use of suitable UV lamp.

Technique # 4. Sterilisation by Filtration: The principal laboratory method used to sterilize solutions of heat liable materials is filtration through filters capable of retaining microorganisms. The pore size of the filters used for filtration is less than 0.75m, which can retain small microorganisms.

Since the diameter of the pores is very small, a suction or a pressure is essential during filtration. However, it is never possible to be certain that filtration procedures, which render a solution bacterium free, will also free it of viruses. There are different types of bacteriological filters used in microbiology. A short description of some of them is given below: 1. Pasteur Chamberland Candle Filter: This type of filter is made of unglazed porcelain. There are different types of this filter depending on their pore size. The grades are L1, L1a, L2, L3, L5, L7, L11, and L13. L1 is the coarser and L13 is the finest in the order. These filters are used for removing organisms from fluid to obtain bacterial toxin. 2. Berkefeld Filter: This is made up of diatomaceous earth pressed in the shape of a candle. On the basis of porosity they are variously classified into V (veal) N (normal), W (dense) etc.

3. Seitz Filter: It consists of a disc of asbestos material through which the fluid is passed during filtration. The disc is inserted into a metal holder before use. After use, the disc is discarded and a fresh one is fitted.

4. Sintered Glass Filter: This is made up of finely ground glass which is subsequently fused to make the small particles adhere to each other. This filter is sterilised after use, avoiding temperature extremes.

5. Cellulose Membrane Filter: There are two types of cellulose filters-Ciradocol membranes (older type) are made up of cellulose nitrate and the modern type is composed of cellulose. Diagrams of some bacterial filters are given in Fig. 1.3.

Culture Media: Majority of bacteria and many eukaryotic microorganisms like algae and fungi can be cultivated under artificial conditions (as opposed to their natural habitats) on suitable culture media. A culture medium must contain all the raw materials that are needed by the particular organism to build up its cellular constituents — carbohydrates, proteins, lipids, nucleic acids etc.

Since water is the most important constituent of all living systems, microorganisms can best thrive in an aqueous medium. The main constituent of the culture medium is, therefore, water, in which the other ingredients are present in a dissolved state.

Microorganisms, like plant cells, are generally invested with a cell wall and they can take up nutrients only when they are in a dissolved state. The major barrier for entry of nutrients into the cell is, however, not the cell wall, but the cytoplasmic membrane. Some of the soluble nutrients can diffuse passively through the membrane, but for the majority there are specific transport systems located in the membrane which help in the uptake of nutrients from the culture medium.

The major elements that are essential for growth of all microorganisms are carbon, nitrogen, hydrogen, oxygen, phosphorus, sulfur, magnesium, calcium, potassium and iron. Many microorganisms also require traces of one or more minor elements, like manganese, molybdenum, zinc, cobalt, nickel, copper, boron, sodium and silicon.

All the elements required for growth have to be provided in the culture medium. Majority of bacteria and all fungi have a heterotrophic mode of nutrition and they are dependent on one or the other organic compound as source of carbon and energy. In general, microorganisms can take up all the other elements in inorganic form.

Thus, for growing the common bacteria and fungi present in soil or water, an inorganic salts medium containing a single organic compound proves adequate. But pathogenic bacteria often refuse to grow in such simple culture media and require supplementation of complex organic compounds, probably because they get accustomed to such compounds while growing in the body of the host. For example, Haemophilus requires haemin and nicotinamide adenine dinucleotide (NAD) in their growth medium.

Although common microorganisms (bacteria and fungi) can grow in an inorganic salt medium with a single carbon-source (generally glucose), they grow much faster when the medium is enriched by addition of some complex organic compounds.

On this basis, culture media can be distinguished into two types: Complex and

Chemically defined.

The complex media contain one or more substances of undefined chemical composition. Commonly employed substances of this type are beef-extract, peptone, tryptone, yeast-extract, malt extract, blood, serum, egg albumin, potato extract, straw-infusion etc.

Casein-hydrolysate is another complex supplement, though its composition is more or less known. A complex bacteriological medium very commonly used for obtaining rapid and good growth is nutrient broth which contains beef-extract, peptone and sodium chloride.

Similarly, for growing saprophytic fungi, a common complex medium is malt-extract agar. Potato- dextrose agar — which contains extract of boiled potato, glucose and some salts — is another medium for growing fungi. Straw-infusion broth is a good complex medium for growth of soil actinomycetes. For growth of pathogenic bacteria blood agar, serum-albumin agar are often used. Among complex media are also to be counted some natural substances like milk, potato, carrot etc.

The chemically defined media, also called synthetic media, contain substances of known chemical composition and each of them is present in a known concentration. A simple synthetic medium which supports growth of common bacteria including Escherichia coli may be prepared from the following ingredients: K2HPO4 7.0, KH2PO4 3.0, Na3-citrate. 3H2O 0.5, MgSO4. 7H20 0.1, (NH4)2SO4 1.0, glucose 2.0, FeSO4 . 7H20 0.01, CaCl2 .2H20 0.01 (g/1). Similarly, for fungi, a synthetic medium is Czapek-Dox. Culture media can be used in liquid form (broth) or in the form of a soft or a relatively hard gel (solid media). Gelatin which was used in the early days of microbiology as gelling agent is no longer in use except for special purposes. It has been completely replaced by agar-agar which is a much superior agent.

Agar-agar is a complex, highly cross-linked polysaccharide extracted from some marine red algae. At a concentration of 1.5-2% (w/v) it produces a solid gel, and at half the above concentration a soft gel. It sets to a gel at 42°-45°C and the gel can be melted at 100°C. The process of gelling and melting can be repeated several times, unless the pH of the medium is acidic.

Unlike gelatin, agar is not hydrolysed by most bacteria. The surface of agar media remains more or less dry, so that colonies appearing on the surface do not spread, and discrete colonies can be easily picked up. All these qualities make agar-agar an ideal agent for solidification of microbiological culture media.

There are some organisms, though very few, like Nitrosomonas which refuse to grow on agar media. For such highly fastidious organisms, the use of an inorganic gel becomes necessary, such as silica gel. It can be prepared by acidifying a solution of sodium silicate with hydrochloric acid. After setting, the gel is washed free of sodium chloride and excess acid. After drying the gel is allowed to absorb sterilized culture medium before inoculation.

Selective Media: When a mixed population of different types of microorganisms is inoculated in a culture medium which allows selective growth of a single type or of a specific group, the medium is considered as selective. Selective media have to be designed according to the necessity for artificially increasing the number of a specific organism or, more commonly, for a specific group of organisms originally present in a mixed population (enrichment) or for direct isolation of a particular type from a mixed population.

A few examples of selective media can be cited. If one intends to study the types of nitrogen- fixing bacteria present in a certain soil, selective medium would contain other ingredients except any nitrogenous compound. In such a medium, organisms which are capable of utilizing atmospheric nitrogen alone would grow.

Again, if one wants to find out the number and select antibiotic resistant strains in a large population, the medium used for the purpose would contain the particular antibiotic at a concentration which is inhibitory for the sensitive strains. The nitrogen-free medium in the first case and the antibiotic- containing medium are examples of selective media.

Differential Media: Whereas a selective medium permits the growth of a selective organism or a selective group of organisms, a differential medium allows growth of various types of organisms present in a mixed population, but at the same time helps to differentiate a particular organism or a group of organisms from the rest.

Thus in a mixed population, the presence of a particular type can be detected. For example, the presence of coliform bacteria in a water sample suspected to be contaminated with fecal matter can be detected by gas production from lactose. The ability to utilize a certain sugar can be similarly tested on a solid medium containing the particular sugar and a mixture of indicator dyes — eosine and methylene blue.

The organisms capable of fermenting the sugar to produce acid form colonies which absorb the dyes and produce a metallic sheen. Thus, on this differential medium (eosine-emthylene blue agar, EMB) the organisms can be visually differentiated from the rest. The endo-agar medium prepared by adding decolorized basic fuchsin in a lactose containing agar medium is another differential medium for identification of coliform bacteria.

9. Enrichment Culture: Enrichment culture technique, developed by Winogradsky and Beijerinck, is based on the Darwinian principle of the survival of the fittest. In an enrichment culture, the environment is preset in such a manner that an organism or a group is selectively encouraged to grow and multiply, so that in a mixed population they become predominant.

The environmental factors that can be utilized for such selective growth or enrichment include carbon, nitrogen and energy sources, oxygen tension, pH, temperature, light etc. These factors have to be selected on the basis of the knowledge about the physiological-biochemical abilities of the organism desired to be enriched.

The question of enrichment arises when the desired organism needs to be isolated from a mixed population in which it forms a minority and is far outnumbered by other organisms. The usual dilution plating method cannot be employed as such, because during dilution the desired organisms are eliminated.

Therefore, before making dilution plates, the number of the desired organisms has to be increased preferentially over the non-desired organisms. In an enrichment culture the competition between the desired and non-desired organisms is removed or minimized, so that after few passages through enrichment culture, the desired organisms become the majority and then they can be isolated by the usual dilution procedure.

Success in such enrichment procedure naturally depends on how effective the selective conditions are. For creating such selective conditions, the knowledge about the desired organism is an essential prerequisite. The enrichment culture technique is a powerful microbiological tool which can be applied for isolation of any specific organism from its natural habitat, provided the selective conditions for its enrichment are known.

A few examples of application of the technique for isolation of different types of microorganisms may be cited: Enrichment of di-nitrogen-fixing bacteria and endospore-forming bacteria: If an inorganic salts medium without any nitrogenous compound and containing an organic compound as carbon and energy source is inoculated with a soil or water sample and incubated under aerobic conditions, the selective conditions thus created will encourage the growth of such organisms which can utilize the atmospheric nitrogen gas as the sole source of nitrogen.

Because the atmospheric nitrogen is in molecular form (N2), the organisms enriched are called di- nitrogen-fixers or diazotrophs. The little amount of combined nitrogen that is present in the soil or the water sample used as inoculum might allow growth of non-fixers at the initial stage, but a few passages through the selective media generally eliminate them. The organisms that can be isolated in this way include different species of Azotobacter, Beijerinckia, Derxia, Azomonas etc. If a similar medium is used and incubated under anaerobic condition, it leads to enrichment of di-nitrogen-fixing anaerobes like Clostridium pastorianum and related species. Enrichment of Clostridia may be further facilitated when the inoculum is pretreated for 5 min at 80°C.

This treatment kills all bacterial vegetative cells, but endospores are spared. On the other hand, a mineral salts-sugar medium with combined nitrogen inoculated with a heat-treated inoculum incubated aerobically is suitable for enrichment of aerobic spore-forming bacteria, mostly different species of Bacillus. By selecting a higher temperature for incubation, it is possible to isolate the thermophilic species of this genus.

The conditions for enrichment of nitrogen fixers and spore-formers are summarised in Table 7.2:

Enrichment of Autotrophic Bacteria: Autotrophic bacteria include the phototrophic and the chemolithotrophic organisms. The phototrophic prokaryotes include the anaerobic photosynthetic bacteria and the aerobic cyanobacteria. The chemolithotrophic bacteria similarly include a variety of organisms capable of oxidizing different inorganic substrates for obtaining energy.

All autotrophic organisms, including green plants, are able to synthesize organic compounds from CO2. For enrichment of autotrophic bacteria of any type, the media must not contain any organic compound, instead there should be a source of CO2, like bicarbonate. For enrichment of all phototrophic organisms, the enrichment cultures must be exposed to light. Cyanobacteria are aerobic and many of them can fix atmospheric molecular nitrogen. For their enrichment a mineral salts medium without combined nitrogen, inoculated with a water or soil sample, should be exposed to light under aerobic conditions.

Cyanobacteria carry out oxygenic photosynthesis like green plants. The other group of phototrophic prokaryotes includes the anaerobic organisms which can be divided into two main types — the photolithotrophs and photo-organotrophs. For both, light is the source of energy for CO2-fixation, but whereas the photolithotrophs can use reduced sulfur compounds like H2S as electron-donor, the photo- organotrophs use simple organic compounds like acetate, malate etc. for this purpose. Conditions for enrichment of different phototrophic prokaryotes are depicted in Table 7.3:

The chemolithotrophic bacteria, like nitrifying, sulfur-oxidising and hydrogen-oxidising bacteria are non-photosynthetic and strictly aerobic. They should be enriched preferably under dark condition to avoid growth of cyanobacteria. The enrichment medium should be composed of inorganic salts including a nitrogenous salt, and an inorganic oxidisable compound acting as energy source.

This compound will depend on the kind of organism to be enriched. For example, for nitrifying bacteria an ammonium salt can be used both as source of nitrogen and of energy. Another group of nitrifying bacteria use nitrite as energy source.

For both types, the enrichment medium is adjusted at an alkaline pH (8.5) and, furthermore, an insoluble acid-neutralizer like CaCO3 or MgCO3 has to be added to counteract the nitrous and nitric acid produced by the bacteria. Carbon dioxide released through interaction of acid and carbonate is helpful for growth. A number of passages through such media are required for adequate enrichment. Another important group of autotrophic bacteria is the sulfur oxidizers. They can grow under autotrophic conditions using reduced sulfur compounds, like thiosulfate, sulfide or elemental sulfur and produce sulfuric acid. For their enrichment, a mineral salts medium including an inorganic nitrogenous compound and the oxidisable sulfur compound is used. The organisms are aerobic and can tolerate extremely acid pH, although they grow optimally at a neutral pH. Some species are obligately autotrophic and some others can grow also heterotrophically (facultative).

A third group of autotrophic bacteria comprises the hydrogen oxidizing organisms which utilize the oxidation of H2 to water as the energy-giving reaction. They are commonly known as hydrogen bacteria and all of them are only facultatively autotrophic. For their enrichment a mineral salts medium with combined nitrogen and without any organic carbon source contained in a closed vessel is generally employed. The gas phase of the vessel is artificially produced by replacing air with a mixture of

H2 (70%), O2 (20%) and CO2 (10%) (v/v). The conditions for enrichment of the nitrifying, sulfur oxidizing and hydrogen bacteria are shown in Table 7.4:

10. Requirements of Macro- and Micro-Elements for Growth: These elements must be provided to microorganisms for growth. Water forms the major part of all actively growing living systems and microorganisms are no exceptions. But microorganisms— particularly endospores produced by some of them — can withstand desiccation for a long time without losing viability. Though, in such a state, their metabolic activities and growth are practically absent.

Besides water, carbon is the most important element that constitutes about 50% of the dry weight of bacterial cells. The importance of this element lies in the fact that carbon forms the skeleton of all organic compounds which are present in all biologically significant molecules, like those of proteins, carbohydrates, lipids, nucleic acids etc.

The majority of microorganisms have a heterotrophic mode of nutrition and they derive their, supply of carbon from one or the other organic compounds, like sugars, organic acids, alcohols, complex carbohydrates etc. All such heterotrophs can also fix small amount of CO2 into/several metabolic intermediates (heterotrophic CO2-fixation). In contrast, the phototrophic and chemolithotrophic microbes draw their carbon requirement fully or for the most part from CO2. Some facultative autotrophs can grow both under lithotrophic (purely inorganic substrates) or under heterotrophic conditions. Some have a mixed type of nutrition, the mixotrophs which can simultaneously utilize CO2 and some organic compound as carbon source. Next to carbon is nitrogen which forms about 10-15% of the dry weight of microbial cells. Most bacteria can grow only in the presence of combined nitrogen supplied in the medium. A few, including some cyanobacteria, can reduce molecular nitrogen to ammonia and incorporate it into organic acids to produce amino acids.

This property, known as di-nitrogen fixation, is conferred by a special enzyme complex known as nitrogenase. Nitrogen is present in proteins, nucleic acids, cell wall polymers, coenzymes, vitamins etc. Among these, proteins are quantitatively the most important accounting for about 50% of the dry weight of bacterial cells. Phosphorus is the next major element forming 2-6% of the dry weight of bacterial cells. Microorganisms get their supply of this element from inorganic phosphates provided in the growth medium. Phosphates also help to keep the pH of the medium in a favourable range by a buffering action.

Among the most important cellular constituents that contain phosphorus are the nucleic acids. DNA accounts for 3-4% of the dry weight of bacterial cells and RNA for 10-20%. The energy-rich compounds, like adenosine triphosphate (ATP), guanosine triphosphate (GTP) etc. and phosphorylated sugars are also among important phosphorus containing compounds. Besides, phospho-lipids form the membrane system.

Sulfur is also an essential element for all living organisms. It is found in all proteins as a constituent of the amino acids, cysteine and methionine. Two cysteine molecules can join by oxidation to form a dimer, called cystine (S-5 bond, disulfide bridge).

This reaction is of special significance in imparting a characteristic folding of the polypeptide chain, and also it plays an important role in joining individual polypeptide chains to produce the quaternary structure of protein molecules. Microorganisms get their supply of sulfur from inorganic sulfates present in medium.

Sulfate is reduced intracellularly by an assimilatory reduction pathway to HS– (sulfhydryl) and incorporated into organic compounds. The element sulfur has special significance in case of sulfur- oxidizing chemolithotrophs, like Thiobacillus and the purple and green photosynthetic sulfur bacteria, like Chromatium and Chlorobium. Some species of Thiobacillus can oxidize elemental sulfur to sulfate and utilize the oxidation energy for chemolithotrophic growth. Purple and green sulfur bacteria utilize sulfide as exogenous electron donor in photosynthesis and in the process they produce elemental sulfur which deposits within or outside their cells.

So far we have considered the role of non-metallic elements in the constitution of microbial cells. Though quantitatively less, metallic elements also form essential parts of microorganisms. Among them, magnesium (Mg++) and potassium (K+) are required in substantial amounts. Mg++ is essential for maintenance of the integrity of ribosomes and it acts as a co-factor in many enzyme reactions, particularly those involving ATP. Moreover, it is present as a part of chlorophyll in all photosynthetic organisms, including cyanobacteria, and purple, green and non-sulfur photosynthetic bacteria containing bacteriochlorophylls. Potassium (K+) is one of the important intra-cellular elements for maintenance of ionic balance. It also serves as a co-factor in many enzyme reactions and it plays an important role in ribosomal function. Iron (Fe++) forms an integral part of all haem-proteins, like cytochromes. It is also present in ferredoxin and nitrogenase. Iron bacteria, like Thiobacillus ferrooxidans, are able to grow chemolithotrophically at the expense of the energy of oxidation of Fe++—>Fe+++. Among other metallic elements required by microorganisms are zinc (Zn++), molybdenum (Mo++), manganese (Mn++), calcium (Ca++), cobalt (Co++) and nickel (Ni++). Zinc and manganese act as cofactors for several enzymes. Molybdenum, together with iron, are integral parts of the enzyme nitrogenase. Another molybdenum enzyme is dimethyl sulfoxide reductase which converts the sulfoxide to dimethyl sulfide. The enzyme has an important role in the sulfur-cycle of nature. Nitrate reductase is still another molybdoenzyme.

Some nitrogen-fixing bacteria possess nitrogenase in which vanadium (Va++) replaces molybdenum. Nickel (Ni++) is required by hydrogen-oxidising bacteria for hydrogenase activity. Some bacteria can ++ synthesize cyanobalamine (Vitamin B12) and they require cobalt (Co ) which is present in the vitamin. Calcium (Ca++) is essential for production of endospores in Gram-positive bacteria. The endospores contain calcium dipicolinate, a compound which is largely responsible for thermo resistance. Zinc and manganese ions serve as co-factors in several enzyme reactions. Sodium ion (Na+) is not generally required by most bacteria, although it is sometimes added in culture media mainly for maintenance of a favourable osmotic pressure of the medium. Most of the common bacteria can tolerate a moderate concentration of NaCl (3-4%), but the marine microorganisms require a higher concentration for growth. There are some extremely salt-tolerant bacteria (the halophiles) like Halobium which need a much higher concentration of NaCl (up to 20%) for maintaining their cell integrity.

Pure Culture of Microorganisms 1. Streak Plate Method: This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium (Fig. 16.13). The successive streaks “thin out” the inoculum sufficiently and the micro- organisms are separated from each other.

It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface.

Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited. Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/needle and re-streaked onto fresh media to ensure purity.

2. Pour Plate Method: This method involves plating of diluted samples mixed with melted agar medium (Fig. 16.14). The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The bacteria and the melted medium are mixed well.

The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.

Pour plate method has certain disadvantages as follows: (i) The picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and

(ii) The microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.

3. Spread Plate Method: In this method (Fig. 16.15), the mixed culture or microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline.

A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod. The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.

The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.

4. Serial Dilution Method: As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media.

A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.

The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms (Fig. 16.16.), i.e., 100 microorganisms/ml of the liquid medium.

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism.

If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.

5. Single Cell Isolation Methods: An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow.

The following two methods are in use: i. Capillary pipette method: Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillars pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture (Fig. 16.17).

ii. Micromanipulator method: Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down.

A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette. Now a hanging drop is searched, which contains only a single microorganism cell.

This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism.

The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialised studies.

6. Enrichment Culture Method: Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture.

The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and, specific condition of incubation favours the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.

Types of Staining 1. Simple Staining: Colouration of microorganisms by applying single dye to a fixed smear is termed simple staining. One covers the fixed smear with stain for specific period, after which this solution is washed off with water and slide blotted dry. Basic dyes like crystal violet, methylene blue and carbolfuchsin are frequently used in simple staining to determine the size, shape and arrangement of prokaryotic cells. (Fig 5.1)

2. Differential Staining: These staining procedures are used to distinguish organisms based on staining properties. They are slightly more elaborate than simple staining techniques that the cells may be exposed to more than one dye or stain, for instance use of Gram staining which divides bacteria into two classes-Gram negative and Gram positive.

3. Gram Staining: It is one of the most important and widely used differential staining techniques in microbiology. This technique was introduced in 1884 by Danish Physician Christian Gram. Gram staining procedure is illustrated in fig 5.2.

In the first step the smear is stained with basic dye crystal violet (Primary stain) followed by treatment with iodine solution functioning as mordant.

Iodine increases the interaction between cell & dye so that cell stains strongly. The smear is next decolourized by washing with ethanol or acetone. This step generates the differential aspect of Gram stains. Gram positive bacteria retain crystal violet and become colourless.

Finally smear is counter-stained with a simple basic dye different in color from Crystal violet. Safranin is the most common counter stain which colours Gram negative bacteria pink to red and leaves Gram positive bacteria dark purple. (Fig 5.2)

The differences in staining responses to the Gram stain can be related to chemical and physical difference of cell walls. The Gram-negative bacterial cell wall is thin, complex multilayered structure and contains relatively high lipid contents in addition to protein and mucopeptide. The higher amount of lipid is readily dissolved by alcohol, resulting information of large pore in the cell wall, thus facilitate leakage of crystal- violet – iodine (CV-I) complex which results in decolorization of the bacterial cell.

Which later take counter stain and appears red. In contrast the cell wall of gram+ve bacteria is thick and chemically simple, composed mainly of mucopeptides. When treated with alcohol, it causes dehydration and closure of cell wall pore, thereby does not allow the loss of (CV-I) complex and cell remain purple.

4. Acid Fast Staining:

It is another important differential staining procedure. It is most commonly used to identify Mycobacterium spp. These bacteria have cell wall with high lipid content such as mycolic acid -a group of branched chain hydroxy lipids, which prevent dyes from readily binding to cells.

They can be stained by Ziehl-Nulsen method, which uses heat and phenol to derive basic fuchsin into the cells. Mycrobacterium spp. were penetrated with basic fuchsin, not easily decolourized by acidified alcohol (acid alcohol) and thus are said to be acid fast.

Non arid fast bacteria are decolourized by arid alcohol and thus are stained blue by methylene blue counter stain.

5. Endospore Staining: Spore formation takes place in some bacterial genera to withstand unfavourable conditions. All bacteria cannot form spores, only few bacterial genera including Bacillus, Clostridium, Desulfotomaculum produce sporulating structure inside vegetative cells called endospore.

Endospore morphology and location vary with species and are valuable for identification Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization.

In the Schaffer-Fulton procedure, endospores are first stained by heating bacteria with malachite green, which is very strong stain that can penetrate endospores. After malachite green treatment, the rest of the cell is washed free of dye with water and is counter-stained with safranin. This technique yields a green endospore with red vegetative cell. (Fig. 5.3)

Preserving Microbial Cultures: Agar Slant Cultures: All microbiology laboratories preserve micro-organisms on agar slant. The slants are incubated for 24hr or more and are then stored in a refrigerator. These cultures are periodically transferred to fresh media. Time intervals at which the transfers are made which varies with the origin and condition of growth.

Agar Slant Culture Covered with Oil (Parafin Method): The agar slants are inoculated and incubated until good growth appears. They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant surface. Transfers are made by removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil, inoculating a fresh medium and then preserving the initial stock culture.

This is a simple and most economical method of preserving bacteria and fungi where they remain viable for several years at room temperature. The layer of paraffin prevents dehydration of the medium and by ensuring an aerobic condition, the microorganism remain in dormant state.

Saline Suspension: Sodium chloride in high concentration is frequently an inhibitor of bacterial growth. Bacteria are suspended in 1% salt solution (sublethal concentration in screw cap tubes to prevent evaporation). The tubes are stored at room temperature. Whenever needed the transfer is made on agar slant.

Preserving Microbial Culture: Preservation at Very Low Temperature: The organisms are suspended in nutrient broth containing 15% glycerol. The suspension is frozen and stored at -15°C to -30°C. The availability of liquid nitrogen (temp -196°C) provides another main preserving stock culture. In this procedure culture are frozen with a protective agent (glycerol or dimethane sulphoxide) in sealed ampoules. The frozen culture are kept in liquid nitrogen refrigerator.

Preservation by Drying in Vacuum: The organisms are dried over calcium chloride in vacuum and are stored in the refrigerator.

Preservation by Freeze Drying (lyophilization): In this process the microbial suspension is placed in small vials. A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or acetone at a temperature of −78oC .The vials are immediately connected to a high vacuum line. This dries the organism while still frozen. Finally, the ampules are sealed off in a vacuum with small flame. These culture can be stored for several years at 40°C. This method is also employed for preservation of toxins, sera, enzymes and other biological material. To revive microbial cultures it is merely necessary to break open the vial aseptically, add a suitable stale medium, and after incubation make further transfers. The process permits the maintenance of longer number of culture without variation in characteristics of the culture and greatly reduces the danger of contamination.

Depending on the chemical constituents from which they are made, their physical nature and their functions, different parameters of media are described here.

Growth of microorganisms

Definition of Growth: In biology, growth is generally defined as an irreversible increase in cellular mass due to active synthesis of all the constituents. Growth results in increase of cell number (except in coenocytic organisms). In multicellular organisms, this increase in cell number leads to increase in size, because the daughter cells remain together.

In contrast, in unicellular organisms, cell multiplication leads to increase in number of individuals in a population, i.e. the population size. So, for bacteria, majority of them being unicellular organisms, growth may be defined as the increase in number of cells in a population. It should be remembered, however, that in bacteria and other unicellular organisms, too, a young daughter cell grows in size before it attains a stage when it can divide to complete a cell-cycle.

2. Measurement of Bacterial Growth: Since bacterial growth results in increase in number and, therefore, in population size, several alternatives are available for its measurement. The cell density i.e. number of cells per unit volume of medium may be determined by counting, by measuring the optical density, by estimating the dry weight or protein, etc. Some of these are direct methods and some are indirect.

One of the obvious ways for measurement of growth of a growing population in culture (cultivation of organisms in the laboratory on media in which they can grow) is their direct counting under the microscope. The number of cells in a given volume of the culture can be counted using specially ruled grooved slides similar to those used by medical technicians for counting blood cells.

The grooved portion of the slide has a known depth and area which is divided into several equal squares. These slides are called counting chambers, different types of which are available, like Petroff-Hausser, Neubauer, Thoma etc. (Fig. 7.1).

A drop of the bacterial suspension is placed on the groove of the slide, covered with a cover slip, lightly pressed to remove excess fluid and the slide is examined under the high-power objective of a phase- contrast microscope. The number of bacteria per small square is counted for fairly good number of squares, averaged, and from the mean the total number of bacteria per ml of the suspension is calculated.

In Petroff-Hausser counting chamber an area of 1 mm2 is divided into 25 equal squares and the depth of the groove is 0.02 mm. If the number of bacteria is too high for counting, the original culture may be suitably diluted and for calculating the final count the dilution factor is taken into account. Motile bacteria are to be immobilized before transferring to the counting chamber. The number of bacteria obtained by the above procedure gives the total count. It is obvious that the total count includes both living as well as dead cells. Living or viable bacteria are capable of producing progeny cells by division.

On a solidified nutrient medium a viable bacterium divides and re-divides to form a large number of progeny cells to form a colony. So, the colony-forming ability is a test for viability. A dead or non- viable bacterium is unable to form a colony under any condition of growth. The number of living bacteria per unit volume of a culture or a population is known as its viable count.

The method followed for determining viable count is based on the principle of serial dilution, first developed by Joseph Lister and later perfected by Robert Koch. There are two variations of the dilution plating technique — the spread plate and the pour-plate methods. In the first method, a measured quantity of a serially diluted sample of the original culture is spread evenly on the surface of the solidified growth medium.

On incubation at an optimum temperature, each viable bacterial cell forms a discrete colony on the surface of the medium. Assuming that each visible colony is the progeny of a single bacterium, the total number of colonies is taken as the number of viable cells present in the quantity of the diluted sample spread on the medium. So, by counting the number of colonies, the viable count can be easily calculated. For reliable results, a good number of replications are necessary.

The method is diagrammatically shown in Fig. 7.2:

[One ml of the bacterial culture containing ~ 107— 109 cells/ml is transferred aseptically with a sterilized pipette to 9 ml of sterilized water or normal saline, mixed thoroughly to get a uniform suspension. Then 1 ml of the 1:10 dilution of the original culture is transferred with a fresh sterile pipette to another tube containing 9 ml of water or normal saline to obtain 1:100 (102) dilution. The process is continued till a dilution of 10-7 is reached. Next 0.1 ml aliquots of the final or last two dilutions are uniformly spread with a sterile bent glass rod over the surface of petridishes containing suitable agar medium. On incubation, colonies of bacteria appear on the plates. Number of colonies on replicate plates is counted, their mean determined and the viable count is calculated, taking into consideration the dilution factor. In the example shown in the figure, the viable count comes to 30.7 x 108 = 3.07 x 109/ml.] For making counting of colonies easier, an instrument called a colony-counter may be used. Essentially, it is a box with a slanting top containing a circular hole of 10-12 cm diameter covered with a frosted glass. There is arrangement for illumination inside the box.

For facilitating counting of small colonies, a magnifying glass of about the same diameter as that of the hole is clamped on a bracket. For counting the petridish is placed in an inverted position on the frosted glass, illuminated from below and observed through the magnifying glass. A marker pen may be used to record the colonies while counting, so that the same colony is not counted more than once.

The second variation of the serial dilution method, the pour-plate technique, is essentially similar to the dilution plating technique, except that the diluted cell suspension is mixed with molten agar medium just before pouring in plates. The temperature should not be so high as to kill bacteria and not so low that agar begins to solidify. Good quality agar sets at a temperature of about 42°C, so 45°-50°C is ideal for pouring. On incubation, colonies appear first on the surface. Gradually, bacteria caught inside the agar also form colonies. These colonies are at first lens-shaped before they emerge on the surface. The number of colonies can be counted as in case of spread plates.

The above two methods can be applied for counting aerobic bacteria only, but not for anaerobic bacteria which are unable to grow under normal oxygen concentration of air. For such organisms the dilution plates have to be incubated under oxygen-free atmosphere.

Common vacuum desiccators in which air is replaced by an inert gas like nitrogen can be used. For highly oxygen-sensitive anaerobes, an oxygen-absorbent like alkaline pyrogallol may have to be additionally used. Also, various types of anaerobic jars are commercially available for this purpose.

A different method for determining viable count is the membrane-filter technique. It is mostly used in case of naturally occurring microbial populations, where the cell density is not high (generally - 106 cells/ml), such as for counting coliform bacteria in water samples. A measured volume of the sample is passed through a sterile membrane filter under negative pressure. The filter should have a pore size less than the diameter of average bacterial cells (less than 1μm). After filtration, the membrane filter disc is aseptically transferred on the surface of suitable sterilized agar medium and incubated till visible colonies appear on the filter surface. The colonies are then counted and the viable count per unit volume of the sample is calculated in the usual way.

An entirely different procedure based on statistical principle is the determination of the most probable number (MPN) of a specific group of microorganisms in natural population. The procedure is generally applied in bacteriological analysis of water samples, but it may be also used for other groups of naturally occurring organisms.

For determination of the MPN of coliform bacteria in a sample of water, aliquots of 10 ml, 1 ml and 0.1 ml of the water sample are inoculated into 5 replicate tubes of lactose broth, each containing an inverted Durham’s tube.

Coliform bacteria ferment lactose to produce gas which collects in the Durham’s tubes.

After incubation for 48 h at 32°C, the tubes are examined for presence of gas, and the number of tubes showing gas accumulation is counted (Fig. 7.3):

The most probable number of coliform bacteria in the water sample is then determined from a standard MPN table (Table 7.1):

While counting bacteria in a population is one way for determination of growth, another parameter — viz. measurement of bacterial mass — can also be utilized. Gravimetric estimation of fresh bacterial cells or of dry cells provides a direct method.

Fresh weight of a bacterial mass can be obtained by collecting the cells through centrifugation, washing them with distilled water to remove the soluble ingredients of the culture medium and transferring the pellet to a pre-weighed container. For determination of dry weight, the washed cell mass is kept at 110°C overnight and weighed. Very careful handling is essential for obtaining accurate result, since the quantity is very small for laboratory-scale cultures. Other methods are also available for measurement of bacterial mass, though they are somewhat indirect. One of these is estimation of total nitrogen of the cell mass by the micro-kjeldahl method. Total nitrogen content of an actively growing culture increases linearly with cell mass.

A similar relationship also exists between total carbon and cell weight. Total carbon content can be estimated by the Van Slyke-Folch method. For routine purpose, estimation of total protein of an aliquot of the culture by any of the common methods gives satisfactory and reliable results.

For total protein, the cells are lysed by treatment with alkali resulting in release of the cell contents, followed by centrifugation to eliminate the cell debris and treating an aliquot with a colouring reagent, like biuret, Folin, Coomassie blue etc.

The colour developed is then measured in a colorimeter. From the colorimeter reading, the quantity of protein (mg/ml) is then read off from a standard curve. A standard curve is prepared using an authentic protein like bovine serum albumin and the same coloring reagent. A standard curve shows known quantities of the protein in one axis and the corresponding colorimetric readings in the other axis.

An indirect but rapid and handy method of estimating cell mass is turbidemetry. Cell mass which is directly proportional to the population density can be accurately measured by this method. As the cell number increases with growth, a bacteriological culture medium turns more and more turbid, allowing less and less light to pass through and at the same time scattering more and more of light by the suspended cells.

For most common bacteria, visible turbidity appears when the cell density is between 106 to 101 cells/ml. When the culture contains less cells, turbidimetric measurement of growth is not feasible. The instrument used for this method is called a photo-electric colorimeter which measures the optical density of a suspension or also the colour density of a coloured solution. The optical density of a bacterial suspension is measured by transferring a portion of a liquid culture to a colorimetric glass tube usually having a diameter (light-path) of 1 cm against a blank (control) which is usually the uninoculated culture medium. A suitable light filter (usually green or blue) is used. Optical density can be measured either in terms of absorbance (A) or transmission (T%).

Generally, a colorimeter has both types of scales. Absorbance is the logarithm of the ratio of intensities of incident light (I0) and transmitted light (1) i.e. A = log (I0/I). It is given in log scale. Transmission percent is the percentage of incident light passing through the suspension and is calibrated in an arithmetic scale. The two scales run obviously antiparallel e.g. the blank has an absorbance of O and T% of 100. As turbidity increases absorbance increases and transmission % decreases. The colorimeter is provided with a light source, some filters for selecting light of a desired range of wave lengths, a colorimeter tube with known light-path, a photo-cell which converts incident light to electric current, a device for amplification of the current so produced and finally a galvanometer for measurement of the electric current (Fig. 7.4). A linear relationship between bacterial cell density and absorbance or transmission exists only when the suspension is relatively thin (Fig. 7.5). Another instrument known as a nephelometer has more or less the same components as a colorimeter, but is more sensitive. It measures the light scattered by the bacterial cells in suspension. The higher the cell density more is the quantity of scattered light.

The apparatus is so designed that it can capture the light rays scattered by the suspended bacteria at right angles to the beam of incident light and convert them into electricity which can then be measured in the usual way.

Both colorimetry and nephelemetry can be used for measurement of growth of only unicellular organisms which form a stable, uniform suspension. If colorimeter reading or nephelometer reading is calibrated against cell number or cell mass, these methods provide reliable means for estimation of growth.

3. Multiplication of Unicellular Bacteria: Majority of bacteria are unicellular organisms and most of them multiply by binary fission which means that each bacterium divides to produce two identical cells. Each of them, after attaining maturity, undergoes similar binary fission to produce two daughter cells. Thus, under ideal conditions, cell number as well as mass double itself after given time intervals depending on the species and on the growth conditions. The time interval between two successive divisions is called the generation time or doubling time. After each unit generation time the number of bacteria doubles. Thus, under optimal conditions, growth of bacteria takes place by geometric progression with a constant factor of 2. Starting from a single bacterium, the increase in number can be represented as 20—>21—>22—>23—>24—>25—>….— >2n after n divisions.

4. Determination of Generation Time: If the number of cells per unit volume of an actively growing bacterial culture at a time to is taken as

N0, then the number at time t during which n divisions have taken place is given by the equation: n Nt = N0.2 , where N, represents the number of bacteria at time t. Expressed logarithmically, the above equation becomes:

It is seen from the last equation that by counting the number of bacteria present in an actively growing culture at time t0 and t, it is possible to determine the number of divisions that has occurred during the time interval between t0 and t. Once this is done, the generation time (g) – which is the time between two successive divisions – can be easily determined, because g = t/n. Again, the number of divisions per hour which is known as the doubling rate (v), becomes v = n/t and also v = 1/g. An example may be given for clarification of the above mathematical considerations.

If an actively growing culture has a cell count of 104/ml at a certain time and which increases to 108/ml after a lapse of 6 hours, then the organism has a doubling rate (v):

And the generation time (g) is

The generation time varies from one species to another and it also depends on the growth conditions. Under optimal conditions, Escherichia coli has a generation time of 20 min which means a single E. coli cell can produce 1,024 cells after 200 min during which 10 divisional cycles have taken place.

Exponential or Logarithmic Growth: When, in a bacterial culture, the population doubles at each generation, the growth is said to be exponential or logarithmic, because the population increases as an exponent (power) of 2 and log2 of the number of cells increases in direct proportion to time. A semi-logarithmic plot of the cell number per unit volume of an exponentially growing population against time gives a straight line (Fig. 7.6). The linear relation between logarithm of cell number and time holds good, only when all the cells in a growing culture are viable. In practice, such an ideal behaviour is hardly expected, because some cells lag behind or become non-viable.

5. Growth Curve: Bacterial growth in a flask — or any other container which can be as big as an industrial fermenter — holding a growth supporting medium is known as a batch culture. The batch culture represents a “closed” system, because the nutrients initially present are gradually consumed during growth- producing metabolic end-products which accumulate in the culture vessel causing change of pH value. There is no provision for addition of fresh nutrients or for removal of end products or adjustment of the pH value which also changes during growth.

When the logarithm of the number of bacteria per unit volume of such a batch culture is plotted against time (hr) beginning with the transfer of some viable organisms to the culture vessel (inoculation) a sigmoid growth curve is obtained (Fig. 7.7).

A typical growth curve shows several distinct phases. These are known as the lag phase, the exponential or logarithmic phase, the stationary phase and the death phase. Sometimes, the later part of lag phase is called the acceleration phase and the early part of the stationary phase is called deceleration phase. The lag phase represents the time interval between inoculation and the onset of the exponential growth. During the lag phase, the cell number does not increase, but individual bacteria grow in size due to active synthesis of cellular ingredients like protein, nucleic acids and carbohydrate.

During the later part of this phase, some of the bacteria begin to divide resulting in a slow rise in total count (acceleration phase). There may be several reasons why the bacteria do not start growing immediately after inoculation in a fresh medium.

If the inoculum is taken from an old culture, or from a culture which was growing in a medium having a different composition, the inoculated cells require some time to adapt themselves in the new environment. So, the lag phase may be considered as an adaptation phase during which the inoculated cells do not remain idle, but they are engaged in synthesizing cell materials as a preparation to initiate active growth.

From the lag phase, the bacteria pass into the exponential phase (logarithmic phase) through the intermediate acceleration phase and they are now fully equipped to initiate growth at a maximum rate. Most cells of the population divide regularly at an interval of each unit generation time resulting in exponential increase of cell number and mass.

However, all cells of the total population do not divide simultaneously, rather they divide asynchronously. As a result, the cell number does not increase in a step-wise manner. The maximal growth rate is maintained throughout the exponential phase and it continues till a point is reached where the population density becomes so high that the growth medium is unable to support further growth.

The growth rate falls and ultimately stops. At this point, the culture enters into the stationary phase. Duration of the exponential phase depends not only on availability of nutrients, but also on other factors, like oxygen supply, pH, temperature etc. and, naturally, also on the generation time. The logarithmic (log) phase cells exhibit their highest activity and, therefore, they are most suitable for measurement of generation time, various biochemical properties and cell size.

As the culture enters into the stationary phase, there is no net increase in cell number, although a majority of the bacteria still remains in a viable state and they continue life activities at the cost of reserve substances stored in the cells.

The duration of this phase is highly variable among different species and may be few hours to several days. From practical point of view, the stationary phase cells are of special significance, because many secondary metabolic products, for example antibiotics, are produced in this phase. Also, for organisms which are cultivated as source of biomass, harvesting in this phase gives maximum yield.

The stationary phase is succeeded by the death phase during which the viable cell count falls exponentially, although total cell count may continue unchanged for quite some time. Thereafter, the total count also falls indicating that the cells undergo lysis and they disappear. Lysis may be due to the activity of their own enzymes (autolysis). It is possible that the cell materials released by lysis may provide nutrition to the still living cells for some time. However, the exact causes of death of bacteria are not clearly understood.

Yield: An important parameter of growth is the yield which can be determined from the growth curve of a batch culture by measuring the dry weight of the total population at the beginning and at the end of the exponential phase. Thus, if Mmax denotes bacterial mass (dry weight, g) at the end of the exponential phase, and M0 the initial mass, then yield M is, M = Mmax – M0 (Fig. 7.8).

Usually, yield is expressed in relation to the substrate consumption as yield coefficient (Y) which is the ratio of yield (dry wt. g) and the quantity (g) of substrate utilized. Conventionally, yield coefficient per mole of substrate consumed is called molar yield coefficient (Ym).

Another form of expression of the yield coefficient is the energy yield coefficient (YATP) which is yield per mole of ATP consumed. For calculating this parameter it is essential to know the pathway of carbohydrate (energy source) breakdown of the particular organism and the quantity (moles) of ATP produced per mole of the carbohydrate. For example, Streptococcus faecalis or Saccharomyces cerevisae breaks down glucose by the EMP (Embden-Meyerhof-Parnas pathway) and, when growing in absence of air, produces 2 moles of ATP/mole of glucose.

While molar yield coefficient (Ym) for both these organisms is 20, i.e. both produce 20 g dry cells per mole of glucose (180 g), energy yield coefficient (YATP) for both is 10. Under aerobic conditions, the yield increases significantly, because of higher energy production by complete oxidation of the substrate.

6. Continuous Culture: The fate of a microbial population growing in a “closed” system like a batch culture is comparable to the fate of a multicellular organism having birth, growth, maturity, and death. The environment in a batch culture is subject to constant changes due to continuous depletion of nutrients, shifting of pH value, partial pressure of dissolved gases, accumulation of toxic metabolites etc.

Due to such changes, the duration of active growth phase is also limited. For many critical experiments, it becomes necessary to maintain a culture in an active state of growth over a prolonged time. This can be achieved by a continuous culture which provides for growth of an organism in a constant environment. A continuous culture represents an “open” system in contrast to a batch culture.

One of the devices for growing microorganisms in a continuous culture is a chemostat developed by Novick and Szilard (1950). In its simplest form, a chemostat consists of a culture vessel provided with an inlet for regulated entry of fresh culture medium, an arrangement for pumping in sterilized air, and an Outlet for maintaining a constant volume of liquid in the culture vessel (Fig. 7.9).

The arrangements in a chemostat ensure a constant regulated flow of fresh medium into the culture vessel and simultaneous removal of equal quantity of culture fluid from the vessel through an outlet device. As a result, a constant volume is always maintained in the vessel.

The system is also provided with an arrangement for pumping in sterilized air through the culture to maintain adequate aeration. In contrast to a batch culture, a continuous culture is more amenable to control. On one hand, the continuous inflow of fresh medium-prevents depletion of essential nutrients and, on the other hand, continuous outflow removes spent medium containing toxic metabolites and bacterial cells. These arrangements ensure a prolonged growth in a chemostat.

The importance of a chemostat culture lies in the fact that the growth rate of an organism can be controlled. This can be achieved by supplying one of the essential nutrients, like carbon and energy source, at a sub-optimal concentration in the inflowing medium. As a result, the organisms present in the culture are starved and are unable to grow at maximum growth rate. At the same time, the inflow rate can be so adjusted that the particular growth-limiting nutrient is immediately and fully consumed by the hungry organisms. The outflow removes continuously a fraction of the population to maintain a constant cell density.

Thus, it becomes possible to establish a steady-state of growth. In a steady-state, the growth rate is constant and it equals the rate of inflow of medium which is known as the dilution rate. The dilution rate is so adjusted that the concentration of the critical nutrient in the chemostat is practically nil, because the nutrient is instantaneously consumed by the starving population.

As a result, the growth rate in the chemostat is maintained at a sub-maximal value. Thus, by regulating the dilution rate, it is practically possible to maintain a steady-state culture almost indefinitely which is in sharp contrast to a batch-culture which passes through different growth phases — ultimately terminating in death.

The relation between substrate concentration and growth rate is closely comparable with the relation between substrate concentration and velocity of enzyme reaction as depicted by the famous Michaelis- Menten equation. The growth rate (μ) increases linearly with substrate (growth limiting nutrient) concentration [s] up to a certain point, and then declines to become flat. The substrate concentration at which the growth rate is half of the maximum rate of growth is designated by Ks (Fig. 7.10).

7. Synchronous Culture: A synchronous culture is one in which all the cells of a particular population are in the same stage of development and they divide simultaneously. In a common batch-culture, the population contains cells in all possible stages of development, some have just been produced by fission, others are in intermediate stages and still others are mature and ready for division.

All these stages are parts of the cell-cycle. For this reason, the cell population of a batch culture divides asynchronously producing a typical exponential growth curve indicating that the logarithms of cell number as well as of cell-mass increase linearly with time (see Fig. 7.6). In a synchronous culture, on the other hand, one would expect a step-wise increase in cell number, because there would be no increase in cell number between two successive divisions. But the cell mass would still show a linear increase with time, similar to that found in a batch culture. This is due to the fact that the newly born cells go on increasing in mass between two successive divisions, though their number does not increase (Fig. 7.11).

Synchronization of cell division in a bacterial population can be achieved in several ways. One of them is repeated alternation of the incubation temperature. A culture growing at the optimal temperature is exposed to a lower temperature, so that the growth is slowed down considerably. After some time, the culture is again brought to its optimal temperature.

The time interval is adjusted according to the growth rate (generation time) of the organism. By lowering the temperature, cell division is delayed and all cells divide simultaneously when the temperature is raised to the optimal level. The treatment has to be continued through several cycles to obtain satisfactory results. Another method of getting a synchronously dividing population of cells is by filtration through a membrane filter.

An asynchronous population of bacteria is filtered whereby the cells are adsorbed in the pores of the membrane filter. In the adsorbed state the cells continue dividing producing progeny cells which are not adsorbed. A reverse flow of sterilized medium through the membrane filter washes away the newly born progeny cells into the filtrate. Thus, a homogeneous population of cells which are approximately at the same stage of development can be obtained. Such cells divide synchronously for a few generations.

The utility of a synchronous culture lies in the fact that the whole culture can be taken as a multicellular complex consisting of individuals having identical development. In contrast, an asynchronous culture can give only an average picture about individual cells. Sometimes, it becomes necessary to gain insight into the sequence of events taking place in individual cells. For smallness of size, study of such events in single bacterial cells is not feasible. A synchronous culture provides this opportunity, because all cells are in the same stage of development. For example, one might want to study the replication of DNA in relation to the cell cycle.

Samples withdrawn at suitable intervals during one unit generation time of a synchronous culture would yield cells at different stages of the cell cycle and each sample would contain cells of a particular stage. A measurement of incorporation of a radioactive DNA precursor would provide useful information of DNA synthesis at different phases of the growth cycle.

11. Physical Factors Influencing Growth: (i) Oxygen: On the basis of oxygen relation, microorganisms are classified into three major types — aerobic, anaerobic and microaerophilic. While aerobic organisms require oxygen of air for growth, anaerobic organisms — which include mainly bacteria — are unable to utilize oxygen. For some obligate anaerobes oxygen is even toxic. Some organisms which are able to grow both in presence of air or in its absence are called facultative anaerobes. The microaerophilic organisms are also aerobic, but they grow only at a reduced oxygen tension.

The aerobic organisms are capable of oxidizing substrates fully to CO2 and H2O using oxygen as the terminal hydrogen acceptor. In this process of respiration they produce ATP in the electron transport system by oxidative phosphorylation from ADP and inorganic phosphate. The obligate anaerobes, on the other hand, can oxidize substrates only partially by fermentation, because of the lack of tricarboxylic acid cycle (TCA cycle)-linked electron transport pathway and they produce ATP only by substrate level phosphorylation.

The facultative anaerobes are essentially of two types. Some are capable of changing their metabolism either to fermentation or to aerobic respiration depending on the environmental conditions i.e. whether oxygen is absent or present.

The other group of facultative anaerobes are respiring organisms capable of utilizing oxygen either from air (when air is present), or from oxidized inorganic compounds, like nitrate or sulphate (when air is absent i.e. under anaerobic conditions).

Under anaerobic conditions they carry out the so-called nitrate or sulphate respiration, in which these compounds serve as the terminal electron acceptor in place of oxygen. They produce ATP by oxidative phosphorylation like aerobic organisms.

Microaerophiles are also oxygen-requiring respiring organism, but they can grow only when the oxygen concentration is considerably lower than normal. This is probably due to the oxygen sensitivity of some of their vital enzymes. In an un-agitated liquid culture these bacteria form a layer below the surface where the oxygen concentration is suitable for their growth. In contrast, the aerobic organisms grow at the surface and the facultative anaerobes tend to accumulate at the bottom, if they grow at all. Bacteria can utilize oxygen dissolved in the growth medium. The solubility of oxygen in water is low and most bacteria are well-adapted to such concentration for normal growth when they grow on agar surface or at the air-liquid interface.

Use of thin layers of culture medium in vessels with a wide surface generally suffices for normal growth of most aerobic organisms. However, when use of larger volumes of medium becomes essential, need for additional arrangement for aeration arises.

For laboratory flask cultures, a mechanical shaker having either a to and fro (reciprocal) or an elliptical movement is commonly used for agitating liquid cultures. When still larger volumes have to be used, arrangements for forced aeration become necessary.

Generally, for this purpose, sterilized air is forced through a sparger producing air-bubbles at the bottom layer of the culture fluid. Larger fermenters are provided with more sophisticated arrangements for aeration. For growing anaerobic organisms, oxygen has to be excluded from the culture medium and the atmosphere existing in the culture vessel. Some anaerobes are somewhat aero-tolerant and they can grow in solid media containing reducing substances like thioglycollate, ascorbic acid or cysteine which presumably minimize the toxic effect of oxygen by interacting with it.

For growing anaerobic organisms in liquid culture, generally closed vessels filled completely with the freshly prepared medium are employed. If an artificial atmosphere (gas-phase) is necessary, it must be oxygen-free. Specially designed containers (anaerobic jars) are available for growing anaerobes.

(ii) Hydrogen-ion concentration: Hydrogen-ion concentration [H+] determines the acidity and alkalinity of the culture medium and is commonly expressed as its pH value which is logarithm of the reciprocal of hydrogen ion concentration (gram molecules per liter). The hydrogen ion concentration of pure water is 1/107 moles per liter corresponding to a pH value of 7.0 (neutral). As [H+] increases pH value falls and acidity increases. Decrease of [H+] from the neutral point results in increase of alkalinity. The relationships are shown in Fig. 7.13:

Most bacteria prefer a neutral to slightly alkaline medium, while fungi and algae grow better at slightly acidic conditions. For each organism, there is a minimum, an optimum and a maximum pH, permitting growth. The total range may be quite broad extending over 2-3 pH units which corresponds to a difference of 100-1,000-fold in hydrogen-ion concentration. In spite of this wide variation, organisms are able to grow because the cell membrane is hardly permeable to H+ or (OH)–. As a result, the cell interior remains more or less neutral. Microorganisms during growth may produce acidic or alkaline substances in considerable quantities to change the pH of the medium to such an extent which proves inhibitory. For example, the nitrifying bacteria produce nitrous and nitric acids as oxidation products and they turn the medium highly acidic and unsuitable for growth. An opposite situation prevails in case of ureolytic or proteolytic bacteria. They produce ammonia as the end-product which turns the medium strongly alkaline.

For checking these extreme and unfavorable changes in the pH of the culture media, they have to be adequately buffered. Generally, the acidic and alkaline phosphates, like KH2P04 and K2HP04, are used in bacteriological media. An equimolar solution of these two salts produces a pH of 6.8 which is suitable for growth of many bacteria. If an organism produces an acid in the medium, it reacts with the alkaline phosphate turning it to the acidic form. Opposite is the case when an organism tends to change the medium alkaline by producing a basic substance. Thus, phosphates which are well tolerated by most bacteria are widely used for buffering. At the same time they serve as source of phosphorus.

Though buffering of the medium is generally found to be adequate for checking pH shifts, sometimes, as in the case of nitrifying bacteria which produce profuse amounts of acids, addition of insoluble neutralizers like calcium or magnesium carbonates becomes necessary.

Bacteria, in general, prefer a neutral to slightly alkaline growth medium. But there are exceptions also. The bacteria growing optimally at an acidic pH are called acidophils and those growing preferentially in an alkaline pH are known as alkalophiles.

Not only bacteria (both eubacteria and archaebacteria) but also a number of algae, fungi or even flagellates are endowed with such properties. Many of the acidophilic organisms are simultaneously thermophilic. Some examples of acidophilic bacteria are Acetobacter acidophilum (optimum pH 3), Thiobacillus thiooxidans (pH range for growth 0.9-4.5), Bacillus acidocaldarius (pH range for growth 2-6, opt. 3), Thennoplasma acidophilum (pH 1-2), Sulfolobus acidocaldarius (pH range 1.5-3.5), Non- bacterial organisms which can grow in an acidic pH include Cyanidium caldarium (eukaryotic alga, pH 2-3), Chlorella ellipsoidea (pH 2), Chlamydomonas acidophila (pH 2), Polytomella caeca (pH 1.4 — a flagellate). Some fungi, like species of Cephalosporium, Trichosporon, Aspergillus, Penicillium and Fusarium, can also grow at considerably acidic media.

The true alkalophilic organisms have pH range for growth varying between 8-11 or even more. They include some blue-green algae e.g. Spirullina and Synechococcus, true bacteria, like Bacillus alkalophilus and several other bacilli and some archaebacteria, like Natronobacterium sp., Methanobacterium thermoalcalophilum etc.

(iii) Temperature: Temperature is one of the most important variables for growth. For every organism exists a temperature range which is congenial for its growth. At one extreme of this range is a minimum temperature where growth can start and at the other extreme is the maximum where growth abruptly stops. In between the two extremes, there is an optimum temperature where growth rate is maximum. On the basis of temperature relations, microorganisms are classically divided into three major groups — psychrophiles, mesophiles, and thermophiles. Psychrophiles are cold-loving organisms growing in marine or alpine habitats. Many of them can multiply at 0°C or even sub-zero temperature and have maximum growth rate between 16°C and 20°C.

Well-known examples of psychrophilic bacteria are marine Photo-bacterium sp. and the iron-oxidizing Gallionella ferruginosa (opt. temp. 6°C). Besides, several marine species of Pseudomonas and some species of Bacillus isolated from glaciers belong to this group.

The majority of bacteria and other microorganisms belong to the mesophilic group having an optimum temperature between 20°-45°C. Growth of most mesophiles stops abruptly between 45°- 50°C. Organisms which can grow at higher temperatures are called thermophiles. Several types of thermophilism can be noticed. Organisms which grow optimally above 65°-70°C and are unable to grow below 40°-45°C are called obligate thermophiles. Organisms which grow at mesophilic temperatures but can survive exposure to 60°-65°C are called thermo-tolerant (e.g. Methylococcus capsulatus).

Organism having a maximum temperature of growth at 50°-65°C, but can grow also at mesophilic temperature are called facultative thermophiles (e.g. Bacillus coagulaus). Among the obligate thermophiles some can grow at temperature ranging between 80°-100°C. They are called hyper- thermophiles. In more or less recent times, a good number of such organisms have been discovered e.g. Thermotoga maritime (max. temp, of growth 90°C), Aquifex pyrophilus (max. 95°C), Desulfurolobus ambivalens (max. 95°C), Pyrobaculum aerophilum (max. 104°C), Pyrococcus furiosus (max. 100°C), Pyrodichtium brockii (110°C), Hyperthermus butylicus (max. 108°C) etc.

Majority of the hyper-thermophiles except the species of the genera Thermotoga and Aquifex are archaebacteria. Interest in the hyper-thermophilic organisms has greatly increased in recent times due to the possible presence of thermo-stable enzymes in them e.g. the thermo-stable DNA polymerase from Thermus aquaticus, technically called Taq-polymerase, is used in PCR.