eBook Chapters

Isolation of Protoplasts

Protoplast is the living material of the cell however an isolated protoplast is the cell from which the  cell wall is removed. In plant breeding programme many desirable combination of characters could not be transmitted through the conventional method of genetic manipulation. Higher plants that could lead to the genetic process involving fusion between the subsequent developments of a product to a hybrid plant is known as somatic hybridization. Plant protoplasts can be isolated from cells by two methods:

Introduction

The actinomycetes are gram positive, non-motile, non-sporulating and nonacid fast bacteria. Sometimes these are called ray fungi and are connecting link between the bacteria and the true fungi. They possess a very fine vegetative mycelium that does not fragment into bacillary or coccoid forms and therefore their colonies on agar are commonly cohesive or tough in consistency. In many soils, the actinomycetes accounts from 10 to 25% of the total count on agar plates. Their number is higher in soils of low moisture content than in wet soils. They are responsible for degrading more resistant carbon material in soil like humus. They are also important flora of manure heaps. Most of these, particularly Streptomyces, produce antibiotics and so are of a great value

Introduction

Soil is the natural habitat of a number of microbes viz. bacteria, fungi, actinomycetes, algae and many viruses. Out of these, bacteria are morphologically very simple as made up of just one cell of small size, therefore can multiply rapidly. Each viable bacterium at appropriate dilution can develop into a colony. Hence by counting the number of these colonies which develop on a selective growth medium, the number of viable cells of bacteria present in soil can be ascertained. Since the number of the bacteria present in soil is very high, the soil suspension is to be sufficiently diluted to reduce the number of colonies and to prevent their overlapping. The number of colonies formed from a diluted sample multiplied by the extent of dilution (dilution factor) gives the number of viable cells present

Introduction

Fungi constitute an important part of microflora of normal soil. In soils, fungal propagules vary from 2 x 10⁴ to 1 x 10⁶ per gram soil. The propagule being considered as fungal hyphae, spore, and hyphal fragments which is capable of giving colony on the plate. Most soil forms have a filamentous organisation capable of reproduction by means of spores. Their development is especially favoured by soils having acidic reactions. Soil fungi has significant role in organic matter decomposition, as a plant pathogens and in the interaction with other soil microorganisms. Agar plate count method is commonly used as a measure of fungal population in soil.

1. Deoxyribo Nucleic Acid (DNA)

DNA is a genetic material, originally confined to nucleus of a cell. In plants, some DNAs are also present in mitochondria and chloroplasts. DNAs are vital macro molecules having high molecular weight.

Isolation of plant DNA is cumbersome or difficult primarily because of the presence of a number of secondary plant products. Secondly, a protocol, which is found suitable for isolation of DNA in one plant, may not work in other. The efficiency of DNA extraction depends on many factors, such as sample material, ionic conditions of the extraction medium and type of lysing agent used etc.

There are two methods, one developed by Dellaporta et al. (1983) and the other by Murray and Thompson (1980) which are most commonly used for extraction of DNA in a number of plant genera.

Murray and Thompson’s (1980) method is described here below with minor modifications.

Special Staining
A. Spore stain
Spores may be spherical or oval and may be central subterminal or terminal in position within the cell. Shape, size and position of the spore in the cell are useful for the identification of sporulated organisms. Spores are difficult to stain and when once stained, they are difficult to decolourize. With Gram’s stain, mature spores remain unstained bodies. Modified Ziehl-Neelsen’s method of staining will be used for the staining of spores.
Composition
a. Concentrated carbol fuchsin
b. Sulphuric acid 0.5 % solution.
c. Loeffler’s alkaline methylene blue
Materials
Old cultures of B. subtilis and Cl. Chauvoei and staining solutions.
Procedure
1. Make smears and fix over flame.
2. Stain with carbol fuchsin and steam for 3 to 4 minutes.
3. Wash with tap water.
4. Decolourize with 0.5% sulphuric acid.
5. Wash with water.
6. Counter stain with Loeffler’s methylene blue for 2 minutes.

The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases.

Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively.

Preliminary Identification of Unknown Bacteria
A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or
dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from
back ground material.

Cell culture is the most convenient and widely used system for the isolation and propagation of many viruses. Either primary cell culture or established cell lines can be used to isolate the suspected viruses. One can normally get 10-100 virus particles per cell inoculated. Many details of the infection process are controllable to a high degree (including cell density, age, multiplicity of infection (M.O.I.), medium composition, cell type and length and temperature of incubation). Many cell types from different species can be susceptible to a particular virus and vice versa. For replication to occur, the cell type used must be permissive for a particular virus under study.

Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA.

Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

1. VARIETAL SEED PRODUCTION

3 m : Rice, soybean

5 m : Blackgram, greengram, cowpea, field lab lab lab lab

5-10 m : Groundnut

25 m : Tomato

Alkaloids are a class of basic, naturally occurring organic compounds that contain at least one nitrogen atom. Alkaloids showed quite diverse medicinal properties (Rates, 2001). Many of them possess local anesthetic properties, but their practical use is limited for clinical purposes (Hesse, 2002). Morphine is one of the most known alkaloids isolated from Papaver somniferum (opium poppy), which had been used and still is for medical purposes. This alkaloid is a powerful narcotic which is used for the relief of pain, but its usefulness is limited because of its addictive properties.

The isolation of microbes is commonly done using serial dilution technique.

1. Trichoderma

Trichoderma is one of the important biocontrol agents. The colonies first produce mycelial growth and then turn yellow and finally produce a dark green color mat.

Anthocyanins are members of the flavonoid group of phytochemicals, a group predominant in teas, honey, wines, fruits, vegetables, nuts, olive oil, cocoa and cereals. The primary players in this group include the anthocyanins (eg, cyanidin, pelargonidin, petunidin), flavonols (quercetin, kaempferol), flavones (luteolin, apigenin), flavanones (myricetin, naringin, hesperetin, naringenin), flavan-3-ols (catechin, epicatechin, gallocatechin), and although sometimes classified separately, the isoflavones (genistein, daidzein) (Skibola and Smith, 2000).

Introduction

The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg^- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme.

Mutagenic Agent

Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10^-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

ABSTRACT

A plant grows in a highly competitive environment. It is continually being threatened by other plants encroaching upon the space from which it draws its sustenance, by microorganisms, by insects, avian, reptilian or mammalian herbivores. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as Spines or Leathery Leaves or Chemical. These defenses are passive, coming into action only when the plant is attacked which repel insects or the herbivores that attempt to feed on them. Plants protect themselves from insect attack by providing a multitude of repellent chemicals including toxic and bitter alkaloids and tannins. Non-specialist insects and other animals that attempt to feed on the plant may be repelled, injured through severe gastrointestinal malfunction or even killed. The cabbage, spinach, mustard etc. are well known for their mustard pungent flavor that deters most animals from consuming it. Even most medicinal plants like Cynanchum collialatum of the family Asclepediaceae have tannins or alkaloids like “cardenolides” that repel most animals and insects. All these plants have a basic enzyme “Myrosinase” which react with these specific tannins to produce the toxic metabolite, which repels insects and animals. But caterpillars of few insects are known to tolerate these toxic alkaloids (ex: caterpillar of Striped Tiger Butterfly-Danaus genutia) and in turn sequester the toxic alkaloids into their tissues making themselves non-palatable to their enemies. We hypothesize the analysis of possible roles of toxic alkaloids isolated from these butterflies as bio-insecticides

1. Mitochondria

Mitochondria are known as the “Power House” of a cell because they are the site of ATP production. In addition, they are also involved with photorespiration and oxidation of fatty acids and work in association with peroxisomes, glyoxysomes and chloroplasts.

Isolation of intact mitochondria is rather difficult. This is because the cell walls of plant tissue are rigid as compared to the single-layered wall surrounding the mitochondria. A significant fraction of mitochondria suffer structural damage during the process of tissue homogenization. Therefore, the pelleted mitochondria need further purification by sucrose gradient centrifugation.

Mitochondria can be isolated from green as well as etiolated leaf; however, the latter is preferred to obtain higher yield of mitochondria (Moore and Proudlove, 1983).

12.1. Routine Media for Fungal Isolation

In recent years commercially available readymade culture media have been used in research laboratories for the isolation of fungal plant pathogens. These culture media are standardized and they are in different types. Some media are used for general purposes while some others are used for the isolation and growth of specific pathogens e.g.

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

Introduction

Mutation refers to any heritable change in the base sequence of a DNA molecule. A mutagen is a physical of chemical agent that causes mutations to occur (or increase the frequency of their occurrence). Mutagenesis is the process of producing a mutation. If the mutation occurs in nature without the addition of a mutagen, it is referred as a spontaneous mutation, and the resulting mutants are spontaneous mutants. If it caused by a mutagen, the process is induced mutagenesis.

Ultraviolet light is a fairly potent mutagen. The main DNA lesions are two chemically different types of covalent dimers: cyclobutane-pyrimidine dimers and pyrimidine dimers. The pyrimidine dimers only account for 20% of the total dimers but are correspondingly more mutagenic. In E. coli the number of mutants induced by ultraviolet (UV) light can be reduced by exposure to visible light (photoreactivation), which implicates cyclobutane pyrimidine dimers since dimers can not be photoreactivated.

Aromatic amino acid of proteins, and purine as well as and pyrimidine bases of nucleic acid absorb ultra violet radiation. Pyridine bases, particularly thymine of DNA is the main target compound involved in bactericidal action of ultra violet radiation. The energy absorbed by thymine causes it to under go a photochemical reaction with adjacent ( adjoining) thymine molecule in same DNA strand. DNA replication is blocked because the formation of thymine: thymine (T:T) dimmers. As the dosage increase, cytosine-thymine (C:T) to (C:C) cytosine-cytosine dimmers can be found to add the lethality of ultra violate radiation.

Background
Milk somatic cells are a diverse population of cells found in milk, including epithelial cells, leukocytes (white blood cells), and macrophages. Isolating these cells from milk provides valuable insights into mammary gland health, mastitis detection, and milk quality assessment.
Several methods can be employed for milk somatic cell isolation. Among the others, centrifugation is the most common method of cell separation. Filtration, immunomagnetic separation and flow cytometry are the other methods of isolating specific cell populations.
In bovines, isolating milk somatic cells is useful in milk quality assessment, mastitis detection, and mammary gland biology research. An increased milk somatic cell count level, particularly neutrophil, indicates mammary gland inflammation. Healthy and mastitis glands can be diagnosed by analyzing the differential counting of milk somatic cells.

Introduction

Biological nitrification is a two-phase process, which consists of the oxidation of ammonia-nitrogen to nitrite-nitrogen by species of genera Nitrosomonas, Nitrosospira and Nitrosogloea, and the subsequent oxidation of nitrite-nitrogen to nitrate-nitrogen by species of the genera Nitrobacter and Nitrocystis. The following equations explain the reaction.The importance of nitrifying organisms in soil is based on their capacity to produce nitrate, the major source of nitrogen assimilated by higher plants. The rate of nitrification is dependent on such factors as concentration of NH₄+, temperature, moisture, aeration and pH. Bacteria have their optimal activity at an alkaline reaction. Below pH 7.0 the process declines.

Background
Peripheral blood mononuclear cells (PBMCs) are a crucial immune system component. Isolating them from whole blood allows for an in-depth study of immune responses, disease mechanisms, and the development of noveltherapies. Lymphocytes, monocytes, and other different  white blood cells (WBCs) possessing circular nuclei make up the cells of PBMCs. These cells can be directly obtained from whole blood using different separating methods. PBMCs can be utilized for multiple purposes, from drug assessment to expression analysis to parentage determination.
• Toxicity assessment of novel compounds: Interaction of drugs with PBMCs can help assess the safety of compounds and set appropriate doses for safe consumption.
• Comparative analysis: In vitro assessment of diseased vs. healthy PBMCs can provide insights into affected biochemical pathways. The effects of foreign chemical compounds can also be assayed to drive future studies.
• Personalized medication: Studying PBMCs can help develop a genetic immune profile, leading to more specificity and effectiveness in personalized medicines.
• Occupational disorders: Diseases developed due to the overexposure of certain toxic compounds can also be determined by testing PBMCs in peripheral blood.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

4.1 From Soil

Soil contains varied group of microorganisms such as fungi, bacteria, actinomycetes, algae and protozoa. The type of nutrients available, moisture, temperature and pH of the soil determine the nature of microbial population in soil. Different laboratory techniques for isolation of microorganisms have been detailed in chapter-III. These are used in particular for the isolation of specific type of microorganisms. All the techniques adopted are just to segregate the pathogens/ microorganisms from the mixture of microbial population present in nature.

(a) Isolation of Fungi

Soil contains large number of fungi and constitutes a major place of microbial population. They can be isolated in pure form by following the methods given below:

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

Introduction

Studies on biocontrol agents have revealed the presence of a diverse range of antifungal metabolites derived from amino acid, polyketones and isoprenoid biosynthetic pathways, each of which has distinctive regulatory points and is subjected to different stimuli within organisms. In this experiment, the extraction of secondary metabolites from the culture of T. viride and Chaetomium globosum will be demonstrated.

RNA is transcriptional product of DNA. The total RNA includes three classes, rRNA, tRNA and mRNA. The ribonucleic acids exists as ribonucleo protein particles in intact cells. The ribonucleo protein complex is dissociated by SDS in to RNA and protein deproutinised by phenol and the free RNA left in the aqueous solution is precipitated in cold by adding alcohol. 

Introduction

Rhizobia are Gram-negative soil bacteria , aerobic nitrogen fixers and adhere to the roots of leguminous plants. Fixed nitrogen is assimilated to proteins and is transported along the plants. A part of proteins is also released in rhizosphere region.

Requirements

1. Yeast Extract Mannitol Agar (YEMA)
2. Sterile saline
3. HgCl₂ solution (1%)
4. Ethanol (70%)
5. Root nodules of Mung bean
6. Water blanks
7. Pipettes
8. Petri plates
9. BOD Incubator

Under natural habitats microorganisms usually grow in mixed populations containing several species. This is really a problem for microbiologists because a single type of microorganism can not be studied adequately in a mixed culture. In laboratories these populations can be separated into pure cultures. The pure cultures contain only one type of organism and are thus suitable for studying the cultural, morphological and biochemical characters of an individual species. In the present days there are several ways of developing pure cultures in laboratories. Some of which are as follows:

Introduction
In order to study the cultural, morphological and physiological characteristics of an individual species, a pure culture of the microorganisms is required. Several different methods of separating the microorganisms from a mixed culture are available. The two most frequently used methods involve making a pour plate and/or streak plate. Isolated colonies are obtained from individual organisms after incubation.
Streak Plate Method
Plating is generally applied to the inoculation of a medium in petridish. Streak plate method is best for the separation of organisms from a mixed culture. In this method, agar medium is allowed to solidify as a thin layer in a petri dish and culture is streaked on the surface of agar medium and allowed to incubate to get isolated colonies.
Materials
1. Sterile petri plate
2. Wire loop
3. Nutrient agar medium
4. Bunsen burner
5. Mixed culture of E.coli and Micrococcus leutus

Isolation of fungal pathogens

a. From leaves

•Choose leaves with young lesions.

•The lesions along with some healthy portion are cut in small pieces.

•The leaf portion is surface sterilized with 0.1% HgCl₂ for 10-15 seconds and washed with sterilized water 4-6 times.

•After washing, infected leaf pieces are placed on PDA plates with a sterilized forcep.

•After two days of incubation at optimum temperature, bits of mycelium from the margins of leaf pieces are transferred to fresh PDA slants.

•In certain cases the leaves are incubated in moist chamber and growth

•from infected portion is transferred directly to culture media.

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

ABSTRACT

Amajor goal of plant biotechnology is to develop effective and lasting means to reduce crop losses due to stress caused by biological organisms. In Indian context, fungal diseases are rated as the most important factor contributing to yield losses. Although, conventional programmes towards development of disease resistant cultivars have been successful , they suf fer from limitations of human health consequences, potential degradation of environment etc. In this scenario, introduction of specific genes encoding for disease resistance through genetic engineering seems to be a viable alternative. Among the various genes that are induced in response to plant defense, chitinases are the most widely studied and used for development of transgenic crops. Keeping this in view, attempt was made to isolate chitinase genes from four fungal species viz., Trichoderma harzianum, Metarhizum anisopliae, Nomuraea rileyi and Alternaria solani by designing primers based on sequence information available in the database. RT-PCR was done using the RNA isolated from the fungal species using the designed primers. Amplified products were eluted, cloned into pTZ57R vector and sequenced. Characterization of sequenced cDNA was done using the BLAST (x) programme of NCBI site. The amplified product (261 bp) from T. harzianum showed 90 per cent homology to endochitinases from T. asperellum and T. viridae. Using this cDNA, full-length chitinase gene of 1556 bp from T. harzianum could be accomplished through RACE (Rapid amplification of cDNA Ends). In M. anisopliae the amplified product of 1500 bp matched the full length gene of 1500 bp and showed 99 per cent homology to M. anisopliae and 96 per cent homology to M. flavoviride. In other two fungal species, viz., Nomuraea rileyi and Alternaria solani, the amplified products did not show any homology to chitinase gene

Mesenchymal stem cells are multipotential cells capable of proliferation and differentiation into osteogenic, chondrogenic and adipogenic lineages both in vitro and in vivo. Minimal criteria for defining MSC according to International Society of Cell Therapy (ISCT) are (a) they must be plastic-adherent when maintained in a standard culture conditions, (b) MSC must express some surface antigen such as CD105, CD73, CD90, CD44 and must not express CD34, CD45, CD 14, CD19, and HLA-DR, and (c) MSC must differentiate in vitro into osteoblast, adipocytes and chondrocytes under specific differentiating condition.

Although traditionally MSC isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including umbilical cord blood, chorionic villi of the placenta, Wharton’s jelly, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, periosteum, deciduous teeth, amniotic fluid, dental pulp, testis, synovium and the circulatory system. Bone marrow and periosteum sources are richest in young animals with their numbers diminishing, but still present in old age. It is estimated that MSCs represent approximately only 0.01 to 0.001% of the total nucleated cells within isolated bone marrow aspirate. The yield of MSCs from bone marrow varies from species to species. e.g., Canine =1 in 2.5 × 10⁴; Feline =1 in 3.8 × 10⁵; Murine =1 in 10.8 ×10³ to 1 in 3.45×10⁴ and Horses =1 in 4.2 × 10³. Despite this low number, they are remains a great interest, as they can be easily isolated from a small aspirate and culture-expanded through as many as 40 population doublings to significant numbers in about 8 to 10 weeks.

Mesenchymal stem cells are multipotential cells capable of proliferation and differentiation into chondrogenic, osteogenic and adipogenic lineages. Minimal criteria for defining MSC according to International Society of Cell Therapy (ISCT) are (a) they must be plastic-adherent when maintained in standard culture conditions, (b) MSC must express some surface antigens such asCD105, CD73, CD90, CD44 and must not express CD34, CD45, CD 14, CD19, and HLA-DR, and (c) MSC must differentiate in vitro into osteoblast, adipocytes and chondrocytes under specific differentiating condition. 
Although MSC is traditionally isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including umbilical cord blood, chorionic villi of the placenta, Wharton’s jelly, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, periosteum, deciduous teeth, amniotic fluid and synovium. Bone marrow and periosteum sources are richest in young animals with their numbers diminishing, but still present in old age. One of the attractive advantage of bone marrow derived mesenchymal stem cells (BM-MSCs) as a source of cell transplantation is their low immunogenecity. Recently, several studies have reported that BM-MSCs may be immune-privileged cells that do not elicit immune response due to an absence of their immunologically relevant cell surface markers. BM-MSCs also are known to inhibit proliferation of T lymphocytes, B lymphocytes, dendritic cells and natural killer cells.

What is DNA

DNA is a simple molecule

DNA is negatively charged

DNA has lots of ring structures; ring structures are generally quite hydrophobic.

DNA is a very robust molecule.

DNA work has lots of toys.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

Plasmid is a double stranded, self replicating, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. The sizes of plasmids range from 1-1000 kbps. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation.

There are various methods available for isolation of high molecular weight genomic DNA from plants. The basic steps involved in all these methods are removal of cell wall, nuclear membrane, proteins, lipids, carbohydrates, cell debris, RNA without affecting the integrity of DNA. The two major factors that affect DNA integrity are action of endogenous nucleases and impact of mechanical shearing.

After precipitation, run-off and surface water storage in lakes and man-made reservoirs, the next steps in the hydrological cycle are infiltration through the unsaturated zone and groundwater dynamics in the saturated zone. Well-established applications of isotopes in groundwater hydrology are

The range of isotope applications in surface water hydrology is not as wide as in groundwater hydrology as the surface waters are more easily amenable to conventional hydrological methods. Following are the areas in which isotopes have a significant role in either improving the accuracy of measurements or in providing additional information, which is otherwise not easily available.

For the first time, isotope dilution analysis was introduced by Hevesy and Hafer in 1934, but it was not until 1940, the usefulness of isotope dilution technique was revived by Rittenberg and Foster (1940). There are three general types of isotope dilution methods. These are (a) direct (b) inverse and (c) double isotope dilution. In general isotope dilution methods may be used for the following type of studies.

    (a)    In isolation purification and identification of unknown intermediate in a chain of metabolic reactions.

    (b)     To obtain evidence of synthesis (incorporation), and precursor-product relationships between known compounds.

    (c)    As an analytical tool in the assay of known compounds. In isotope dilution techniques, the labelled intermediate, which is being diluted by unlabelled carrier, is a known compound.

2.1 Isotopes

In 1913, J.J. Thomson and Aston introduced atmospheric neon into the positive ray apparatus and got two parabolas corresponding to masses 20 and 22, with the intensities of the tracks corresponding to the ratio of 9:1, irrespective of the condition of discharge and pressure. This indicated that neon existed in two forms having atomic weights 20 and 22, and at the same time it could not be distinguished chemically. These two types are known as isotopes of neon (iso = same, topas = place in the periodic table). When the average atomic weight of the ordinary neon was calculated based on the abundance of these two forms in the ordinary neon, it was found to be 20.2 which agreed the chemical methods. Thus the existence of isotopes of a given element was discovered.

Formerly isotopes were defined chemical properties but with different atomic weights, the same atomic number and occupying the same position in the periodic classification. Later this definition was slightly modified and now isotopes are defined as nuclides with the same number of protons but differing in the number of neutrons, for example, ordinary hydrogen (also called protium ; proton =1 and neutron =0), deuterium (heavy hydrogen ; proton =1 and neutron = 1), and tritum super heavy hydrogen (proton =1 and neutron =2). Investigations using mass spectrographs have revealed more than 280 isotopes which are stable artificially radioactive isotopes.

4.1 Selection of Isotopes for Tracer Studies

In radioisotopes we have all the essentials of a powerful research tool, a non-destructive means by which atoms could be followed and detected in the organism. Application mainly depends on the fact that chemical properties of the isotope of an element are essentially identical.

The basic requirement of a tracer is that it be

1.Chemically and physically, virtually equivalent to the substance it represents or displaces and that it, in no appreciable way, affect the system differing from its normal counterpart.

2.Radioactivity could be detected directly and

3.Unaffected by the physical or chemical treatments that are given. Hence, this has been used widely in the investigations of the various problems in biology, chemistry, physics and other fields like agriculture.

1. INTRODUCTION

The contribution of Extension and research to Israel’s agricultural productivity and profitability is a collaborative effort with research hailing back to the late 19^th century. It facilitated technological innovation in agriculture, transfer of information, training of farmers and connectivity. These were in effect a dictate of continuously deteriorating agricultural terms of trade, crippling water shortages and severe climate and soil constraints.

The Israeli Extension Service’s (IES) adoption of Information and Communication Technologies (ICTs) significantly enhanced extension’s capabilities and scope of activities. ICT empowered extension’s ability to develop, introduce and adopt innovative agricultural technologies and collaborate with research and other partners. Adoption of ICT as one of the tools of technological innovation dramatically improved the transfer and management of information, production chain efficiencies and integration within and with the agricultural sector. These were and are critical success factors contributing to Israeli Extension, Research, Services and Farmers ability to sustain a profitable and thriving agriculture sector. As such the overall results are a major contribution to rural viability and an illuminating case study of technological innovation adoption.