eChapters

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

The spermosphere represents a short-lived, rapidly changing, and microbiologically dynamic zone of soil surrounding a germinating seed. It is analogous to the rhizosphere, being established largely by the carbon compounds released into the soil once the seed begins to hydrate. These seed exudations drive the microbial activities that take place in the spermosphere, many of which can have long-lasting impacts on plant growth and development as well as on plant health.

Generally microorganisms are so small and colourless in their natural state that it becomes difficult to observe them clearly under microscope. Stains and dyes, which are colouring agents, are used by biologist for better understanding of the morphological and anatomical structures of the organisms under study. Stains are prepared with great care and specification to get satisfactory result, while dyes are not as exacting in their characteristics.

Dyes are of two types, natural and synthetic. The natural dyes are used for histological purposes, whereas the synthetic dyes for bacterial stain. Usually dyes which are derivatives of cyclic compound benzene of benzole, are prepared from one or more substances found in coal-tar, and so are called coal-tar dyes. Chemically dyes are organic compounds containing both chromophore and auxochrome groups linked to benzene rings. Chromophore group provides the compounds the property of colour and thus benzene containing chromophores are called chromogens. It has no affinity to combine with fibres or tissue and as such not called dye. For being a dye, compound should contain an auxillary group which provides the compounds the properties of electrolytic dissociation.

The dyes may be acidic, basic or neutral. The acidic dyes are eosin, acid fuchsine and picric acid whereas the basic dyes are safranin, methylene blue, crystal violet etc. The acidic dyes ionize to give the dye portion of molecule a negative electrical charge. They are salts of sodium, potassium, calcium, magnesium etc. The basic dyes ionize to give the dye portion of molecule a positive electrical charge and are salts of chlorides, sulphates or acetates.

Abstract

Alleviating plant salt stress and remediating saline soils are of great economic interest. Beneficial microbes such as arbuscular mycorrhizal (AM) fungi, plant growth promoting rhizobacteria (PGPR), etc. are associated with many plant species including trees. These beneficial microbes can cope up with salinity and help the plants to survive in such soils. Diversity status of beneficial microbes from the samples collected from different salt affected areas was investigated by many researchers. They screened and identified the efficient PGPR isolates isolated from saline areas for plant growth hormone (IAA) production and phosphate solubilization. The significance and highlights of the recent research as well as the past understanding of beneficial microbes and their role in reclamation of saline areas is discussed in this paper.

Introduction
Sterilization of culture media, containers, and instruments is crucial in bacteriological work to ensure the isolation and maintenance of pure cultures. Sterilization and sanitization are essential processes in a microbiology lab to prevent contamination and ensure accurate results. Sterilization refers to the complete elimination of all microorganisms, including bacteria, fungi, viruses, and spores, using methods like autoclaving, dry heat, filtration, and chemical agents. Sanitization, on the other hand, reduces microbial load to a safe level through cleaning and disinfection using alcohols, detergents, and UV light. These practices maintain aseptic conditions, protect lab personnel, and preserve sample integrity. Proper sterilization and sanitization are fundamental to microbiological research, clinical diagnostics, and industrial applications, ensuring reliable and reproducible experimental outcomes.
Preparations for Sterilization Before sterilization, cleaned and dried test tubes and flasks should be carefully plugged with clean, non-absorbent cotton. Pipettes should also be plugged at
the mouth end, and the plugs should remain in place during use to minimize oral contamination. Cotton plugs provide sufficient ventilation for bacterial growth while preventing external contamination.

Introduction

Microbes like all other living beings require suitable nutrients as well as favourable environment like pH, osmotic pressure, temperature, humidity etc. for growth. The substrate/food base which fulfills any of these requirements and supports the growth of an organism is called culture medium. Selection of a medium to grow a particular type of microorganism is extremely important, as the microbes vary in their nutritional requirements. Thus, different media are used for different groups of microbes. The following are the main types of media:

a) Natural/Empirical media: These are made up of natural substrates, such as herbaceous or woody stems, seeds, leaves, corn meal, wheat germ, and oatmeal etc.The exact chemical composition of this media is not known. e.g. Nutrient agar

b) Semi-synthetic media: 

Introduction
Microbiology techniques are used for various objectives like:
• To observe and isolate the micro-flora
• To test the microbiological quality of various food and dairy products
• To find the presence of any particular pathogenic microorganism
• To maintain a pure or mixed culture of specific microorganisms
1. Serial Dilution Techniques
• Bacteria are often present in such huge numbers that they can be difficult to count. Yet accurate counts are necessary for a variety of reasons like, for instance, assessing the quality of water or the safety of food. There are a number of counting techniques but most rely on the dilution of the sample to reduce the bacterial numbers down to a quantity that can be counted accurately.
• All bacterial plate count methods require the sample to be serially diluted until an estimated count of 30-300 colony forming units (CFU) on the plate is obtained. Plates with more than 300 CFU are very difficult to count. Plates with less than 30 CFU are not statistically reliable.
• If turbidity is observed in a broth culture, it might have millions more bacteria than you need. If this turbid broth is used directly for plating, all the CFU would grow together into a confluent mass and we would not be able to distinguish one colony from another.

Introduction
A) Cultivation of Fungi
A fungus is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, which is separate from the other eukaryotic life kingdoms of plants and animals. A characteristic that places fungi in a different kingdom from plants, bacteria, and some protests is chitin in their cell walls. Similar to animals, fungi are heterotrophy; they acquire their food by absorbing dissolved molecules, typically by secreting digestive enzymes into their environment. Fungi do not photosynthesize. Growth is their means of mobility, except for spores (a few of which are flagellated), which may travel through the air or water. Fungi are the principal decomposers in ecological systems. These and other differences place fungi in a single group of related organisms, named the Eumycota (true fungi or Eumycetes), which share a common ancestor (form a monophyleticgroup), an interpretation that is also strongly supported by molecular phylogenetics. This fungal group is distinct from the structurally similar myxomycetes (slime molds) and oomycetes (water molds). The discipline of biology devoted to the study of fungi is known as mycology. In the past, mycology was regarded as a branch of botany, although now known fungi are genetically more closely related to animals than to plants.

Introduction

A microscope is an optical instrument consisting of a lens or a combination of lenses for making magnified images of minute objects. A simple microscope consists of a single lens of magnified glass held in a frame, usually adjustable and has a stand for holding the object and a mirror for reflecting the light. But a compound microscope consists of two lenses i.e., objective and eye piece respectively. A compound microscope gives much greater magnification than simple microscope. The compound microscope will be extensively used in this course in studying various morphological and staining characters of soil microorganisms.

Our exposure to wide variety of chemicals has increased markedly in the recent past. Most of these compounds are mutagenic as well as potential carcinogens. Emphasis is being laid to develop method for detection of environmental carcinogens so that their use can be avoided.

Despite the fact that mammalian cell structure and human enzymatic pathways differ from those in bacteria, the chemical nature of DNA is common to all organisms; this permits the use of bacterial test systems for rapid detection of possible mutagens.

The Ames test which was developed by Bruce Ames in 1970, at the University of California, U.S.A. is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the amino acid histidine biosynthesis operon.

S. typhimurium, the indicator organism, used in this test is the mutant strain that cannot synthesize histidine (His^-), lack the enzyme to repair DNA so that mutation shows up readily and has leaky cell wall that permit the rapid entry of chemicals. In addition, this S. typhimurium strain carries mutation enhanced plasmid, the combined effect of these characters increase the sensitivity of the organism to mutagenic treatments.

(A) Effect of Temperature
Introduction
Temperature is one of the most important physical factors affecting microorganisms. Bacteria are different from higher plants and animals in the lack of homeostatic mechanism and cannot regulate heat generated by metabolism and are, therefore, directly and readily affected by temperature. Over a limited temperature range, there is a two-fold increase in the rate of enzyme catalyzed reaction for every 10°C rise in temperature.
Bacteria may be divided into three major groups with respect to their temperature requirements: (i) Psychrophiles, those with optimum temperature between 0°C and 20°C; (ii) Mesophiles, with optimum temperature between 20ºC and 40°C and (iii) Thermophiles, with optimum temperature between 40°C and 80°C. Thermophiles are of two types: (a) Facultative thermophiles with an optimum temperature of growth between 45°C and 60°C and (b) Obligate thermophiles with optimum temperature above 60°C. Temperatures in the range between 50°C and 100°C are normally in the lethal range for bacterial cells and spores. The range of temperature preferred by bacteria is genetically determined, resulting in enzymes with different temperature requirements.
Each organism grows within a particular temperature range (i.e. cardinal temperature points). The minimum growth temperature is the lowest temperature at which growth of a species will occur. The highest temperature at which a species can grow is its maximum growth temperature, and a species grows fastest at its optimum growth temperature.  Time of exposure is a vital factor in assessing the

Introduction
Sugarcane (Saccharum officinarum Linn.) is a remarkable tall, perennial grass characterized by its thick, jointed stalks and long linear leaves. This remarkable plant, known for its ability to efficiently photosynthesize in warm climates, is a C4 plant. The propagation of sugarcane primarily occurs through stem cuttings, as it seldom produces flowers and seeds. It finds roots in regions with tropical and subtropical climates featuring warm temperatures, abundant sunshine, and well-drained fertile soil (Shukla et al., 2017). Cultivated for its sweet juice, rich in sucrose, sugarcane serves as the primary raw material for the production of sugar, ethanol, and various other valuable products. Its lifecycle comprises several growth stages, including germination, tillering, grand growth, and maturation, culminating in the harvesting of its valuable juice. Continuous advancements in botany and genetics have contributed to the improvement of sugarcane cultivation, enhancing yield and resistance to pests and diseases. The origins and domestication of sugarcane can be traced back to Southeast Asia, particularly in regions encompassing Papua New Guinea, Indonesia, and their vicinity. The domestication of sugarcane is believed to have commenced thousands of years ago when early human populations in these regions recognized the sweetness of the cane and began cultivating it for its edible properties. Over time, through selective breeding and propagation of plants with desirable traits, these early cultivators played a pivotal role in shaping the sugarcane we know today. This indigenous Southeast Asian plant spread to other parts of the world through trade and exploration. Historical records indicate that sugarcane found its way to India around 500 BC and reached China in the 6th century AD. Subsequently, it traversed the Middle East, the Mediterranean region, and eventually reached the Americas during the early voyages of exploration and colonization (Grivet et al., 2004). Christopher Columbus played a significant role in introducing sugarcane to the Caribbean islands during his second voyage in 1493, and from there, it made its way to other parts of the New World, including Brazil and various South American countries. The cultivation of sugarcane emerged as an essential industry in these regions, profoundly influencing their economic and social landscapes. The domestication and widespread cultivation of sugarcane have left an indelible mark on the global economy, trade, and human history. Today, sugarcane remains one of the most significant crops globally, with major producing countries, including Brazil, India, China, Thailand, and others. It stands not only as a pivotal

Microbial transformations of sulfur (S) are even more complex than those of nitrogen because of the large number of oxidation states of S and the fact that several transformations of S also occur abiotically. Although a number of oxidation states of S are possible, only three are significant in nature, 2 (sulfhydryl, R–SH, and sulfide, HS-), 0 (elemental sulfur, S0), and 6 (sulfate, SO₄ 2-). The bulk of S Earth’s S is in sediments and rocks in the form of sulfate minerals, primarily gypsum (CaSO₄) and sulfide minerals (pyrite, FeS2), but the oceans constitute the most significant reservoir of SO₄ ^2- in the biosphere. A significant amount of S, in particular sulfur dioxide (SO₂), enters the S cycle from human activities, primarily the burning of fossil fuels.

Hydrogen Sulfide and Sulfate Reduction

A major volatile S gas is hydrogen sulfide (H₂S). Hydrogen sulfide is produced from bacterial sulfate reduction or is emitted from sulfide springs and volcanoes. Although H₂S is volatile, different forms exist depending on pH: H₂S predominates below pH 7 and the nonvolatile HS- and S^2- predominate above pH 7. Collectively,H₂S, HS, and S₂ are referred to as “sulfide.” Sulfate-reducing bacteria are a large and highly diverse group and are widespread in nature. However, in anoxic habitats such as freshwater sediments and many soils, sulfate reduction is SO₄ ^2- limited. Moreover, because organic electron donors (or H₂, which is a product of the fermentation of organic compounds) are needed to support sulfate reduction, it only occurs where significant amounts of organic material are present.

Sulphur in nature follows similar cycle as that of nitrogen and shares many common features with it. In soils, decomposition of organic matter releases major portion of nitrogen and sulphur bound in proteins, as cystine or methionine amino acids, or vitamins, etc. In proteins, on an average, for every 36 atoms of nitrogen one atom of sulphur is present. Mineralization supplies a large fraction of nitrogen and sulphur to the plants. Secondly, both nitrogen and sulphur have several chemical forms under different soil conditions (Table 8.1) and both are subject to microbial reduction. Thirdly, both can enter or leave soil in gaseous form and both are subject to some degree of leaching in the anionic form.

Present work was aimed to determine the antibacterial activity and minimum inhibitory concentration (MIC) of active crude extracts of selected cyanobacterial species. Nine cyanobacterial strains viz. Anabaena variabilis, A. fertilissima, Nostoc muscorum, N. punctiforme, N. linckia, N. commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. belonging to family; Nostocaceae, Oscillatoriaceae and Stigonemataceae were selected from the herbarium of Indian Agricultural Research Institute, New Delhi, and maintained in BG-11 growth medium throughout the study in the laboratory.

Introduction 
Sorghum bicolor L. Moench, commonly known as sorghum, has emerged as a significant and promising candidate for both sugar and lignocellulosic biofuel production. Cultivated sorghum varieties exhibit a wide range of phenotypic and morphological characteristics, and based on their intended use, they have been categorized into four distinct groups: grain, forage, energy, and sweet sorghum. Grain sorghum varieties typically stand between three to six feet in height, featuring large ear heads. Their primary role is to serve as a source of food for humans or livestock feed. These varieties play a crucial role in providing sustenance and nutrition to both people and animals. Sweet sorghum, also known as sweet stalk sorghum, represents a unique group of genotypes that accumulate soluble sugars within their stalks. These varieties can grow impressively tall, reaching up to twenty feet, and produce significantly higher biomass yields compared to grain sorghum (Murray et al., 2009). Sweet sorghum’s stems are characterized by their thickness and fleshiness, although they do tend to have relatively lower seed yields. Despite this, sweet sorghum offers substantial advantages in terms of bioethanol production due to the high sucrose content present in its stalks (Srinivasa Rao et al. 2009). It has garnered recognition as an ideal candidate for bioenergy crop cultivation in tropical and temperate regions around the world, particularly in the southern United States. When compared to other bioenergy crops such as corn, wheat, sugarcane, sugar beet, cassava, and sweet potatoes, sweet sorghum presents several noteworthy benefits (Disasa et al., 2016). It is notably drought-tolerant and requires significantly less water than many other crops, such as one-third of the water needed for sugarcane and half of that required for corn. Furthermore, it exhibits tolerance to salinity, enabling its cultivation in marginal regions that are not traditionally suitable for crop production. Additionally, sweet sorghum boasts lower greenhouse gas emissions on a life-cycle basis, contributing to a more sustainable and environmentally friendly biofuel production process. The applications of sweet sorghum are diverse, with its stalks being used for various purposes. In Brazil and India, the stalk is chewed fresh or processed for the production of food-grade syrup and alcohol. Its high sucrose content makes it an excellent candidate for bioethanol production, with the potential to yield up to 8,000 liters per hectare, surpassing the ethanol yield of corn by approximately twofold and outperforming