eChapters

Of all kinds of resources; financial, natural, physical and human resources, the most critical resources for any endeavor are the human resources involved in it. Physical, natural and economic resources are important for facilitating development of any nation but the most important are the human resources. Human resources are the most significant of all the resources to convert all other available resources into production and economic entities. Capable, efficient and motivated human resources can bring about the tremendous positive change towards progress. No doubt, it is equally true that inefficient people can wither away a whole empire and squander away its precious wealth and abundance. People are the real wealth of nations.

Resource appraisal is very significant for micro-enterprise development especially in rural areas. A developing economy like India needs entrepreneurs who are competent to perceive new opportunities and are willing to incur the necessary risks in exploiting them. Rural areas are infrastructurally poor and to take up any entrepreneurial venture in such a scenario appears to be enormous to the simple farmer who has been engaged in cultivating the land in a traditional manner. Developing agri-enterprises is needed urgently to enhance the productivity and incomes of the farmers from their ever shrinking agricultural lands.

Introduction

National Commodity & Derivatives Exchange Limited (NCDEX) is a professionally managed on-line multi commodity exchange offering commodity futures trading in a wide range of commodities viz, agricultural, base metals, precious metals, energy, ferrous metal, and others. The shareholders of NCDEX comprises of large national level institutions, large public sector bank and companies. Promoter shareholders of the Exchange include ICICI Bank Limited (ICICI), Life Insurance Corporation of India (LIC), National Bank for Agriculture and Rural Development (NABARD) and National Stock Exchange of India Limited (NSE). Other shareholders are: Canara Bank, Punjab National Bank (PNB), CRISIL Limited, Indian Farmers Fertiliser Cooperative Limited (IFFCO), Goldman Sachs, Intercontinental Exchange (ICE) and Shree Renuka Sugars Limited. NCDEX is the only commodity exchange in the country promoted by national level institutions. This unique parentage enables it to offer a bouquet of benefits, which are currently in short supply in the commodity markets. The institutional promoters and shareholders of NCDEX are prominent players in their respective fields and bring with them institutional building experience, trust, nationwide reach, technology and risk management skills. 

A. Answer the following questions:

1.    What is Basis of scientific feeding?

Ans: Knowledge of the quantitative needs of the body for various nutrients and the relative value of feeds as sources of them is the basis of scientific feeding.

2.    What is the minimum requirement of animal per group to conduct a feeding trial?

Ans: Minimumanimal per group should be 4.

3.    When you will go for t test and ANOVA test?

Ans: In case of two rations, ‘t’ test and if it is 3 or more then ‘Analysis of Variance’ or ‘ANOVA’ test is applied.

4.    What are the advantages of feeding trials with laboratory animals?

Ans: It involves much less cost of animals, less feed and labour cost,requiredlesstime,individualvariabilitycanbe minimized due to similar genetic and nutritional history which is not possible in case of large animals, large number of animals can be used at a time and easy to slaughter the animals for histological and chemical analysis

5.    What is the purified form of nutrients?

Ans: Casein, urea or purified soybean protein for protein; starch, glucose or sucrose for carbohydrate; oils or lards as source of fat; chemically pure salts as minerals; pure crystalline compounds as vitamins.

A. Write True or False of the following statements

1.    Goats have higher threshold level for bitter taste than other ruminants.

2.    Cattle is acknowledged as mobile pruning machine.

3.    Creep feed should have at least 10% DCP and 58% TDN. 4. Goat under brows condition performs better.

5.    Colostrumshould be fed to the kids @ 10ml/kgbodyweight.

6.    Creepfeed maybe introduced to kidsfrom101 days onwards.

7.    Due to difference in MBS, DMI is higher in larger breeds than smaller breeds.

8.    30-40% of feed intake by goat consists of leaves of shrubs and bushes.

A. Answer the following questions

1.    What are metabolic diseases?

Ans: It is defined as disturbances of the internal homeostasis of the body brought about by an abnormal change in the rate of one or more critical metabolic processes.

2.    How do you prevent milk fever by nutritional management?

Ans: By providing a ration that contains 0.5-0.7% Ca and 0.3-0.4% P and by feeding 20 Million Units of vitamin D/cow/ day starting about 5 days before calving and continue for maximum7 days postpartum.

3.    What is the nutritional management strategy to prevent grass tetany?

Ans: A salt lick of 10 parts each of MgSO₄ and Ca(PO₄)₂ with 80 parts of salt is effective. Besides a mixture of 2 parts of MgO and 1 part salt is also effective measure.

4.    Write down the preventive measures of ketosis?

A. Answer the following questions

1.    Why Oats are mostly preferred by horse?

Ans: Because oats are low in energy, rich in fibre (8-11%), very soft and easily ruptured duringmastication, highly palatable, rich in oil content (5%), better protein quality (high lysine), more readily digested in the S.I. than maize and barley and moreover it provides a more open textured material in the stomach due to high fibre and are thus least likely to cause colic compared to other cereals.

2.    Why wheat is most likely cereal to cause colic in horse?

Ans: Because of its high gluten content since gluten leads to the formation of doughy lump in the stomach. Hence wheat should not be used exceptin small quantities or in low levels.

3.    Why Excessive feeding of wheat bran leads to ‘Big head disease’ or ‘Millers disease’ in horse?

Ans: It is due to an imbalance ratio of Ca and P in this bran i.e high in P and low in Ca. So, due to a Ca deficiency, Ca is withdrawn from the bone and the bone is replaced by a proliferationof fibrous connective tissue whichismainlyfound in the skull or jaw.

Nutrition involves various bio-chemical and physiological activities which transformfeed elements into bodyelements. These feed elements are nutrients which are digested, absorbed, utilized to build and renew the components of the animal body. As a result, animal grows and produces-milk, eggs, wool with the help of energy so produced in the body. Afterweaning, most of our farmanimals obtainall of their feedsupply from plants.Barringcarnivores, theplant kingdom is the original and essential source of all animal life, because plants are able to utilize the energy of the sun to build substances which nourishthe animal. Plants make use of carbon dioxide, water and mineral salts to form carbohydrates, fats and proteins which are utilized in the life processes of animal body. Thus plants store and animals dissipate energy.

Plants and their parts

Animal body and plants contain the same substances but they differ in relative amounts. There are also much larger compositional differences among species than do animals. Water is the principal constituent of living plants as well as animal body. With the maturity of seed, water content decreases. The dry matter of plants consists principally of carbohydrates, which serve as both structural and reserve material, while in animals, protein comprises the structure of the soft tissues and fat is the reserve. Thus, animal body contains only a trace of carbohydrate, though occurring as much less than 1 per cent at any given moment, because it is constantly beingformed and broken down in metablolism. Whereas, this is the principal constituent of most of the plant species. Protein is the main constituent of active tissues and thus leaves are much richer in this nutrient than are stems. Grass hays contain less protein than leguminous hays. When theplant matures, there isgradualmovementof protein fromthe vegetative parts to the seed. Thus, the seed contains a higher percentage of protein than the rest of the plant, as in grains and stovers.

Mixture of nutrientscomprisingcarbohydrates, fats, proteins, minerals, vitamins and water are feed stuffs. From the intestinal wall, very few of such nutrients can pass directly to the body tissues, therefore they must be degraded down into simple compounds before they pass through the mucous membrane of the gastrointestinal tract (G.I.T.) intothe bloodand lymph. This degradation process is regarded as ‘digestion’. Thereafter, the movement of these simple compounds through the mucous membrane oftheG.I.Tract into the blood iscalled absorption. Metabolism is the utilization of these nutrients by the animal body and then excretion of the end products takes place from the body. The digestion of feeds is carried out by three means namely, mechanical action: mastication in the mouth and the muscular contraction of G.I.Tract; chemical action: this is carried out by enzymes secreted by digestive juices and microbial action: this is also enzymatic action, but the enzymes are secreted by microbes instead of host.

In ruminants, it is the rumen which harbours bacteria, protozoa and fungi (Fig. 1) ; whereas in non-ruminants, it is the large intestine (Fig. 2). There are four compartments in the stomach-reticulum, omasum, rumenand abomasums. Reticulumprovides ruminantadditionalspacefor storage.Also, if byanychance, foreign bodies like nails, wires etc. reach over here alongwith feed, they are retained for longer periods and thus, soft organs are protected. Omasum squeezes out water from the feed with the help of its strong muscular walls. In the suckling calves or lambs, the two compartments-rumen and its continuation reticulum are less developed and both are joined by a tube like structure -oesophageal or reticular groove, which directly opens into abomasum. That is why in these sucklings, milk reaches straight into the abomasum, which is the true stomach. Rumen develops at about six weeks of age in these young ones. As these sucklings begin to eat solid feed, both rumen and reticulum enlarge greatly and separate. Now oesophageal or reticular groove does not exist.

Thousands andthousands ofsamples ofvarious feeds andfoddersweresubjected to Weende analysis fortheir chemical composition have been depictedin various manuals. Such proximate principles of these feeds provide only the potential value forreference or consultationpurpose, but not theactualvalue of feedstuffs. Losses of nutrientsinfaeces(solids), urine (liquid), gasesetc.duringthe digestion, absorption andmetabolismof theanimal shouldalso be taken into consideration. Therefore, a need arises to conduct digestibilitytrial or experiment for the feeds in the ruminant animals. Major portion of the nutrients present in the feeds are not properly digested in the gastrointestinal tract (G.I.T.), hence not completely available to the animal body. Digestibility is that portion of a feed or any single nutrient of feed, which is not excreted in the faeces or dung and it is therefore, assumed to be absorbed by the animal. When this digestibility is expressed in percentage, it is termed as ‘digestibility coefficient (D.C.)’.

Crude protein of feedscontain true proteinandnon-proteinnitrogen. Trueprotein is madeupaminoacids andforthemaximumefficiency, feed musthave essential amino acids in correct proportion, correct balance as well as non essential amino acids should also be in sufficient amounts. About digestion in simple stomached animals, the true protein is degraded to oligo peptides (less than ten peptides) in the stomach, subsequently to mono peptide and amino acids in the small intestine. Thereafter amino acids are assimilated in the small intestine. But in ruminants, the situation is complex in the sense that feed proteins get digested in the rumen but even amino acids are also broken down by microbes and thereafter, amino and carboxylic groups are released. Secondly, synthesis of new amino acids or proteins takes place for the formation of their own microbial body coat. Because of these reasons, the approach for protein evaluationor expressionisdifferent inruminant andnon-ruminant animal.Further, non-protein nitrogen portion of feed can not be utilized effectively by non – ruminants like swine and poultry. These animals are usually fed with oil cakes, cereals and cereal by-products, which are poor in non-protein nitrogen compounds, however, young succulent fodders, clover etc. are rich in such compounds.

Declaration of facts about the amounts of nutrients required by the various classes of livestockfor differentphysiologicalfunctions like maintenance, growth, lactation, eggproduction, wool productionetc. are in generaltermed as “Feeding standards”. Nutrient requirement is the statement of what animals on an average require for a particular function.Allowanceis greater than the required amount by a ‘safety margin’ which is allowed for variations in requirement between individual animals. Such standards may be expressed in quantities or in dietary proportions, for example gram per day, this method of expression is used mainly for animals given exact quantities of feeds; or gram per kilogram of the diet, this method is used for animals fed to appetite.

Various units are used for feeding standards – for ruminants, the energy requirements may be stated in terms of net energy, metabolizable energy or feed units and protein requirements in terms of crude protein, digestible crude protein or metabolizable protein. It is obviously desirable that the units used in the standards should be the same as those used in the evaluation of feeds. For dairy cows, the requirements are given separately for maintenance and for milk production. But for growing chickens, they are given for maintenance and growth combined. In some instances, the requirements for single functions are not known, for example, vitamins and trace elements. As mentioned above, in feeding practice, the requirement amount is accompanied by the addition of a ‘safety factor’.

Dog Feeding

In Asia, dogs are the main pet animal kept both by urban and rural families. They are very versatile and adopt to common food habbits of their masters. Most often, they are fed on the home cooked foods – bread, rice, vegetables, pulses, milk and non – vegetarian meals. Many processed pet foods are also available in the market in the form of “Dry biscuits” or in “Canned form”, which provide all the requirements for Energy, Protein, Vitamins, Minerals and some moisture. Like any other farm animal, dogs also require these nutrients for their maintenance and productive purposes like – Growth, Lactation, Gestation, draft [sledge – cart on the ice] etcetra. Dogs and humans have been companions from time immemorial and the first readymade dog food was marketed under the name - DOG CHOW CHEKER in the year 1926. Since then such dog – foods have gained popularity. Most of the dog foods are manufactured abroad and marketed in India by Multi – National Companies (MNCs). Following are the guide – lines for feeding :

1. Energysources: Cereal grains - Jowar, Bajra, Oat, Maize, Sorghum.Average requirement of energy 3000 k.cal / kg of feed.

2. Protein sources: Vegetative origin : verypoor in Essential Amino acids. e.g. Soybean, grams, peas [green proteins], beans, other legumes.

3. Fat: 10 % (oil seeds are good sources Protein as well as Fats).

4. Non – starch fibrous feeds (5 %): To tone – up Alimentary canal.

5. Feedsupplements:Additional nutrients:Greenvegetables (carotenoids), fruits, skimmed milk, curd, butter – milk.

6. Feed Additives: These are Non-nutrients: Mushroom, herbal preparations [no side effects by Aanwala, Aloe-veera].

Feeding wild animals in captivity presents a fearful challenge. There arehundreds of species to consider, each representing millions of years of evolutionary adaptation to particular ecological system.

Natural foods range from Ants to Antelopes. (Antelope = African (deer like) ruminant, forexample-gazelles, gnus, kudus and impala) and Mouth structures varyfromEdentate (toothless)toCarnassial(specialized pre-molars and molars).

Stomach : These may be

a) Single

b) Quadruple.

Intestines: These may be

a) Tube likeor

b) Equipped with a caecal fermentation vat (vat = large vessel).

On the surface, the problem appears unsolvable, … it certainly is not easy. But the need persists to nourish all captive species as appropriately as possible … it is not feasible to wait until a DATABASE is developed comparable with that which exists for domestic animals.Actually domestic animal data can be very useful for the estimation of nutrient needs of wild counterparts.

Sincehundreds ofyears, Livestockanimals arean integralpart ofcrop–livestock system. In the life style of Indian people, livestock had always been given a special and respectful place and this will continue in the years to come. Despite considerable deficit of feeds, the above may be one of the reasons of steadily increasing livestock production in our country. Almost all assessments made during the past eight decades, have shown deficits of different orders in respect of following for the availability of :

• Dry fodders

• Green fodders and

• Concentrates

Such surprising phenomenon is inviting our attention for reviewing systems of assessment for finding out the status of consumption and the gap between the above. Production of milk, meat, fibre, chicken and eggis increasing, though. Therefore, there is gap between the availability and requirements of different feeds. Because India is facing shortage of both protein and energy rich animal feeds, a large number of agro – forest based unconventional by products have been used. Some of these feeds were found to be promising, while others affected the performance of the animals, due to presence of incriminatingfactors present in them. But after detoxification, these feeds were suitable for animal feeding. Some of these feeds are :

Gas Chromatography (GC) was discovered in 1954, a verysensitive technique, whichpavedwaytothisworldfor the resolution ofthe sampleleadingtodetection … identification of the minutest quantity of the desired sample.

Fattyacids were usuallyconverted into correspondingmethyl esters (FAME) to improve volatility and to reduce peak tailing for GC. Usually, FAME could be conveniently prepared by heating lipids with a large excess of either acid- or base-catalyzed reagents. Whereas, acid-catalyzed reagents form FAME from both free fatty acids (FFA) and O-acyl lipids base-catalyzed reagents caused only transesterification, i.e. conversion of O-acyl lipids to FAME and do not usually form FAME from FFAs. A base-catalyzed reaction was a useful alternative to acid-catalyzedreactions whena lipid sample contained acid-labile FAs such as propenoids, present in seed oils from the Malvales order, such as cottonseedoil. To effect solublizationof nonpolarlipids such as triacylglycerols in methanol. the predominant solvent in derivatization reagents, benzene was frequentlyadded to the derivatizalion reaction mixture. However, due to health concerns, use of benzene was discouraged. To help solubilize nonpolar lipids, methylene chloride and tetrahydrofuran had been used in place of benzene.

This procedure allows us to mix two feed stuffs with different nutrient concentrations and come up with a mixture of the desired total concentration. For this square to work, the desired diet nutrient concentration must be between the nutrient concentrations of the two feed stuffs as well as in the same unit. The procedure can be used for Crude Protein, Energy, Minerals and so on and so forth.Also, in place of percentage, calories, parts per million/ billion / trillion and other units of measurement can be used.

Double Pearson Square Method

Sometimes, we might want to have the exact amounts of two major nutrients such as - Crude Protein and Energy.

For example, suppose we want a final mixture with 12 % Crude Protein (CP) and 74 % Total Digestible Nutrient (T.D.N.). We have :

Introduction

First feed mill was established in 1875 A.D. in the state of Illinois, U.S.A. for calf meal production.

First feed mill in India was established in Ludhiana, Punjab in 1969 A.D.

Historical Aspect

The primitive man during his long history as a hunter and food gatherer, used ruminants as food till about 6000 B.C. Later, he developed the arts of aerable -agriculture and animal husbandry. The ruminants played a major role as sources ofbothfoodandmechanicalwork. In turn, the new techniquesof foodproduction greatlyincreased the amount of food available and enabledthe establishment of settled communities.Theradicalnatureof thesedevelopments was clearlyshown by the fact that within a couple of millennia, manhad evolved from primitive to urbanized, civilized communities. Throughout the historical epochs, animal food - meat, milk and milk products continued to form an important fraction of the diet in most of the civilized societies. The favoured nature of these foods was well shown in the observation that, with increasing affluence, human -beings tended to increase the fraction of the diet provided by animal foods, especiallyfromruminants.

Since middle of twentieth century, evidences had been accumulating to indicate that the high intake of saturated fats from meat and ‘dairy products might play a role in the etiology of atherosclerosis. This concern had provided a stimulus to produce husbandrytechniquesleadingto the modification of the composition of ruminant fats as well as possibility of protecting dietary poly - unsaturated fatty acids (PUFA) from ruminal biohydrogenation in 1951. Scientists all the world round, started studying ruiminant fats and ruminal lipid metabolism in this perspective. Different research groups developed various techniques of protectingdietary- PUFA, so as to make themavailable in the meat, milk and its products, but lacked the availability of sensitive techniques for confirmation. With the discovery of Gas Chromatoaraphy (GC) in 1954, a new beginning of resolution and detection of PUFA started with gradual precision. Later on, the GC was further equipped with mass - spectroscopy and subsequently with infra -red detector. The scientists were very much successful in protecting the dietary essential fatty acids (E.F.A.s) or even by - passing them from biohydrogenation in vitro.

Plant physiology is a sub-discipline of botany concerned with the functioning, or physiology of plants. Closely related fields include plant morphology (structure of plants), plant ecology (interactions with the environment), phytochemistry (biochemistry of plants), cell biology, genetic, biophysics and molecular biology. Fundamental processes such as photosynthesis, respiration, plant nutrition, plant hormone functions, tropism, nastic movements, photoperiodism, photo-morphogenesis, circadian rhythms, environmental stress physiology, seed germination, dormancy, stomata function and transpiration, both parts of plant water relations, are studied by plant physiologists. The field of plant physiology includes the study of all the internal activities of plants those chemical and physical processes associated with life as they occur in plants. This includes study at many levels of scale of size and time. At the smallest scale are molecular interactions of photosynthesis and internal diffusion of water, minerals, and nutrients. Major sub disciplines of plant physiology include phytochemistry (the study of the biochemistry of plants) and phytopathology (the study of disease in plants). The scope of plant physiology as a discipline may be divided into several major areas of research.

Viruses are much simpler than cells

Protein capsid

Genetic information stored on DNA or RNA:

Types of genome:

Double strand DNA: sometimes circular

Single strand DNA: sometimes circular

Double strand RNA

Single strand RNA

Some can be crystallized

What is an IVR

IVR or Interactive Voice Response is a technology that automates interaction of a database with telephone callers. Enterprises are increasingly turning to IVR to reduce the cost of common sales, service, collections, inquiry and support calls to and from their company.

IVR solutions are generally used with pre-recorded voice prompts and menus to present information and options to callers, and touch-tone telephone keypad entry to gather responses. Modern IVR solutions however, also enable input and responses to be gathered via spoken words with voice recognition.

IVR solutions enable users to retrieve information including bank balances, flight schedules, product details, order status, movie show times, and more from any telephone. Additionally, IVR solutions are increasingly used to place outbound calls to deliver or gather information for appointments, past due bills, and other time critical events and activities.

This book is designed to introduce to the students some of the most widely used experimental procedures in biotechnology, including DNA/RNA isolation, manipulation, cloning, protein purification and characterization. The students will also get familiarized with some of the equipments frequently used in biochemistry and molecular biology.

The biotechnology laboratory course is an exploration of procedures. Hence, in order to get full benefit from the course, the students need to read the manual, which is self explanatory. The more effort they put into the course work, the more they will learn. Before the start of actual lab works, one needs to spend some time reading the Laboratory Manual. This reading will provide background information and an outline of the procedures to be performed.

The biotechnology laboratory is conducted as a “directed” research project. This means that although the general procedures are well established, the overall goal of each experiment is the acquisition of new information. Because of the nature of scientific research, predicting the outcome of experiments that have not previously been performed is difficult. It may therefore be necessary to design new experiments based on the results of previous ones, or to repeat experiments that yielded uninterruptable or ambiguous results.

In molecular biology and biochemistry, the ability to accurately and reproducibly measure and transfer small volumes of liquids is critical for obtaining useful results. For volumes less than 1 ml, the most common method for measuring liquid volumes involves the use of a device known as a pipetman or micro-pipette.

The pipetmen can be both fixed and variable volumes. Commonly used pipetmen are: P1000, P200, P20, P10 etc. P1000 are useful for volumes from 200 to 1000 µl. P200 are useful for volumes from 20 to 200 µl. P20 are useful for volumes from 0.5 to 20 µl. For beginners fixed volume pipetmen are more useful. It is important to ascertain whether the correct pipetman is chosen for measuring the volume one needs.

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Cell Genotypes

A large variety of bacterial strains, like E. coli, are frequently used in molecular biological experiments. Students should choose the strain or strains required for an experiment on the basis of genetic properties of the strains. It is thus pertinent that a sound knowledge of the genetic makeup of the E. coli stains is acquired.

The genetic makeup of the strains of E.coli is distinctly different from the wild type (K12 or B strains) strains from which they are derived and contains genes that can be modified. Genes are supposed to be mutated when they are not listed in a genotype. In most cases, the genes listed are mutated to the point of inactivity. In a few cases, a “-” (indicating inactive or absent or deleted), “+” (indicating active). , The genotype is a factor in choosing a bacterial strain. Chosen strains should be ascertained for their utility for the specific experiment.

Small Scale Cultures

Fresh culture from a freshly streaked plate should be taken while experimenting with E. coli cells. For preparing a small scale culture of E. coli , prepare 3-5 ml of LB medium/broth (or appropriate broth include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. Inoculate one tube with a single colony from a fresh plate. The second tube is used as a broth control. Incubate both tubes at 37°C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Never allow cells to overgrow. For some applications, cells can be stored at 4°C for short periods prior to use.

Principle

Apart from PCR for generating large amounts of DNA, the second method is to use bacteria to replicate the DNA and then purify the replicated DNA from the bacteria. Plasmid DNA is usually the DNA of interest for this method. A plasmid is a double stranded circular DNA molecule that will replicate in an organism. A typical plasmid contains four important features:

The plasmid must be circular, because bacteria generally will not replicate linear ? DNA.

The plasmid must contain a sequence that functions as an origin of replication (ori).

The plasmid must contain a selection mechanism that will force the bacteria to retain the DNA; the most common type of selection mechanism used in bacteria is a gene for resistance to an antibiotic such as ampicillin or tetracycline.

The plasmid must contain a region for the insertion of the experimental DNA. A generic plasmid exhibiting these features is shown at right.

An expression plasmid that allows expression of foreign DNA should have a strong promoter segment for transcription of inserted DNA.

Must have an effective ribosome binding site that will allow efficient translation of the transcribed RNA.

Must have high copy number, i.e., the host bacterium should have many copies (usually >100)

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. One can perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a plasmid “mini-prep,” yields fairly clean DNA quickly and easily.

Procedure

Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.

Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.

Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

Solution Preparation

1. Glycerol mix (1 L)

Weigh 25 grams (liquid weight) of glycerol and

Add dH₂O to 1 Liter (Autoclave).

2. Potassium mix (1 L)

170 mL of 1M KH₂PO₄ (monobasic),

720 mL of 1M K₂HPO₄ (dibasic), and

110 mL of dH₂O (Autoclave)

Preparation of Solutions

GTE buffer

50 mM Glucose,

25 mM Tris-Cl,

10 mM EDTA,

pH 8

NaOH/SDS lysis solution

0.2 M NaOH,

1% SDS

Reagents

EDTA

Ethanol, absolute

Isopropanol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)

Sodium chloride (NaCl)

Sodium Dodecyl Sulfate (SDS ), 10%

Tris EDTA (TE), pH 7.4

Tris EDTA (TE), pH 8.0

Tris HCl, pH 8.0

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Isoamyl alcohol

Phenol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

RNAse A

Sodium dodecyl sulfate (SDS) solution, 10%

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Is amyl alcohol

Phenol

Proteinase K

RNase A

Sodium acetate, pH 5.2

Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M

TE Buffer (Tris-EDTA), pH 7.4

Tween 20

Xylene

Principle

Restriction digestion is one of the most common reactions performed in molecular biology. Restriction endonucleases (RE) are also known as restriction enzymes, which recognize short, specific DNA sequences. There are three types of RE: Type-I, Type-II and Type-III. Type II RE, most commonly used for DNA analysis and genetic engineering, cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. All Type II RE recognises a unique nucleotide sequence at which it cuts a DNA molecule and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences. Many RE will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation.

REs can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or “blunt end”. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called “cohesive ends”, as these single-stranded overhangs could potentially come together again through complementary base-pairing, as shown below:

Principle

Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated. This restriction enzyme digest will create a pattern of specific DNA fragments that will serves as a “DNA ladder”.

Principle of DNA Ligase Mechanism

The process of connecting two pieces of DNA together is called ligation, and is catalyzed by an enzyme called ligase. The ligase used most often in molecular biology is derived from the T4 bacteriophage, and uses ATP to supply the energy necessary for the reaction. In addition to ATP, T4 ligase requires DNA with a 5´-phosphate group and free 3´-hydroxyl groups.

Applications

Ligation of blunt or cohesive-ended DNA fragments.

Repair of nicks in double-stranded nucleic acids.

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

Principle

Micropropagation is especially useful for include propagation of high value plants that grow slowly (bulbs, ferns, orchids, palms, etc), new cultivars having limited stock and high market demand (new cultivars produced via conventional breeding or genetic engineering), cultivars that are difficult to root by conventional techniques, and endangered plant species. In addition, virus elimination is possible via culture initiation from meristem explants. Also, micropropagation provides a source of sterile tissues that can be used for other tissue culture protocols, including genetic transformation.

Micropropagation is possible using multiple types of cultures; however, shoot cultures are typically employed, in which case five stages to the micropropagation process are typically recognized as outlined below. Nodal explants of rose are used for culture initiation. Three media, differing in hormone content, is employed and the explants should be evaluated for differences in axillary shoot growth and proliferation.

Microbial contamination sources: Foods may be contaminated by microorganisms at any time during harvest, storage, processing, distribution, handling, or preparation. The primary sources of microbial contamination are soil, air, animal feed, animal hides and intestines, plant surfaces, sewage, and food processing machinery or utensils. Since most foods either carry or eventually acquire bacteria, molds, or yeasts, microorganisms are the major causes of food spoilage. The increased load of microorganisms leads to rapid spoilage.

Organisms: Bacteria and fungi (yeasts and molds) are the principal types of microorganisms that cause food spoilage and food- borne illnesses. The typical microorganisms that cause food spoilage are bacteria (e.g., Pseudomonas), yeasts (e.g., Candida), and molds (e.g., Rhizopus). The growth of microorganisms in the food will increase the load of microorganisms, and the heavier load will add to the difficulty of preventing spoilage and may influence the kind of spoilage to be anticipated.

Food spoilage may be defined as any change that renders food unfit for human consumption. These changes may be caused by various factors, including contamination by microorganisms, infestation by insects, or degradation by endogenous enzymes (those present naturally in the food). In addition, physical and chemical changes, such as the tearing of plant or animal tissues or the oxidation of certain constituents of food, may promote food spoilage. Foods obtained from plant or animal sources begin to spoil soon after harvest or slaughter. The enzymes contained in the cells of plant and animal tissues may be released as a result of any mechanical damage inflicted during postharvest handling. These enzymes begin to break down the cellular material. The chemical reactions catalyzed by the enzymes result in the degradation of food quality, such as the development of off- flavours, the deterioration of texture, and the loss of nutrients. The enzymatic changes in foods are desirable to some extent as in the case of ripening of fruits, but continued enzymatic activity beyond an optimum point becomes food deterioration due to rotting. The native enzymes may be inactivated by heat, radiation or by the use of specific chemicals.

10.1 CEREALS AND CEREAL PRODUCTS

CONTAMINATION: The sources of contamination in cereals and cereal products are soil, insects, air, etc. Most of the microorganisms are removed with the outer portions of the grain during milling. The bleaching agents used during milling also reduce numbers of microorganisms. The bacteria in wheat flour include Bacillus, Micrococcus, Flavobacterium. Mold spores are chiefly of Aspergillus spp and Penicillium spp and in addition Alternaria, Cladosporium and other genera are found. The numbers of microbes in cereals and cereal products vary widely from few hundred per gram to million. Corn meal contains several hundred to several thousand bacteria and mold per gram.

Cultural characteristics of bacteria deal with the appearance of the growth in various liquid and on solid media. The growth characteristics of most of the bacteria are typical of the group or species and help in their identification. The terminology employed to describe various features of bacterial growth are mentioned below.

When unwanted organism settle on objects, substances or pure culture is called contamination.

Infection

Invasion of the body of a man or an animal or a plant by disease producing organism. Infectious disease is a condition brought about by an infection of the body by an organisms which is responsible for the disease. Organism that are capable of bringing about infectious diseases are called pathogens while those organism without this capacity are said to be non pathogenic.

Commensals

Organisms on skin and mucous membrane which multiply without causing damage to the tissue.

Subclinical infection / latent infection

When organism enters the tissue and lead to immunity without the appearance of any definite clinical symptoms.

Abstract

The Adam bridge, the curvilinear chains of islands linking India (Thanushkodi) with Sri Lanka (Thalaimannar), has been a matter of scientific and mythological debate. While the great epic Ramayana says that Lord Rama built this Adam bridge around 1.5 million years ago to bring back his wife Seetha from Srilanka who was a captive by king Ravana of Srilanka, some reports gave another view that it is coral islands of 1.7 million years of age. Under this backdrop, the author refutes the claim made by NASA that the Adam bridge was located firstly by one of its satellites and cited evidences that the Indian scientists located and mapped it using the IRS satellite data much earlier. Further, on the basis of bundles of beach ridges dated in Tiruthuraipoondi-Vedaranniyam area and as well as some of these continue up to Uchipulli area in Ramanathpuram district, the author assigned an age of 3500 YBP ( Years Before Present) to Uchipulli beach ridges seen in the main land to the west of Rameshwaram and Adam’s bridge and inferred that the Adam’s bridge must be equal to or younger than 3500 YBP. The easterly nosing of Indian mainland in Ramanathapuram – Rameshwaram – Thanushkodi and its further extension in the form of Adam bridge, the author demonstrated that the because of anticlockwisely rotating littoral currents to the north and clockwisely moving littoral currents to the south of Adam bridge, the chains sandy islands namely Adam’s bridge was formed by the segregation of sands in between currents shadow zone after 3500 YBP.

Studies and Findings

There was some coverage in the newspapers some time ago about the discovery of a land bridge (Adam’s Bridge) between Dhanushkodi (India) and Talaimannar (Sri Lanka) by NASA through one of its satellite mission. It was also written in some articles that this land bridge was constructed by Lord Rama 1.7 million years ago to reach Sri Lanka with the aim to bring back his wife Sita from the clutches of Ravana. However, this linkage was refuted by NASA. It accepted that the picture was captured by one of its satellites, but it was unaware of the linkage of the land bridge with the great Indian epic Ramayana. Some reports gave another view and mentioned that the bridge was nothing, but 1.7 million years old coral reefs. Many historians have also come out against linking the Adam’s Bridge with Ramayana.

Abstract

Historically, the tiny island katchatheevu located in between Rameshwaram and Srilanka belongs to Ramnad Kingdom and it was handed over to Britishers and during the British’s rule. During that time both Indian and Srilankan fishermen communities were using this for their fishing related activities. On a goodwill, in 1994, the Government of India donated this island to Srilanka. Since then it has become a major issue and a perpetual problem to Indian fishermen that they could neither do fishing around nor berth their boats for drying their fishing nets. Under this scenario, an attempt was made to evaluate the geological setting of Katchatheevu island using E-Topo data which provides the sea bottom topography/morphology. The study revealed that the Katchatheevu island is the north eastern extension of the Pisasumunai of the Rameshwaram island and such arcuate nature of the land between the Pisasumunai and Katchatheevu is attributed to anticlockwisly spiralling littoral currents which would have dumped the sands and developed the island. Thus the study brings some newer information that the Katchatheevu is geologically a part of Indian land mass.

Studies and Findings

The boundary problems within the nations and amongst the nations, especially amongst the territorial nations, have started emerging as major issues of global concern. Katchatheevu Theevu located between India and Sri Lanka is one such a major issue, as the said island which originally belonged to Ramnad kingdom was handed over to Sri Lanka by India in 1974 and since then, the Indian fishermen community is facing untold miseries in the hands of Sri Lankans and hence it warrents immediate attention.

Validity : The degree to which a measurement actually measures or detects what it is supposed to measure.

Value added : This usually refers to firms, where it is defined as the value of the firm’s output minus the value of all its inputs purchased from other firms. It is therefore a measure of the profit earned by a particular firm plus the wages it has paid. As a rule, the more value a firm can add to a product, the more successful it will be. In many countries, the main form of indirect indirect taxation is value-added tax, which is levied on the value created at each stage of production. However, it is paid, ultimately, by whoever consumes the finished product. Another definition of value added refers to the change in the overall economic value of a company. This takes into account changes in the combined value of its shares, assets, debt and other liabilities. Part of the pay of company bosses is often linked to how much economic value is added to the company under their management.

Value at risk : Value at risk models, widely used for risk management by banks and other financial institutions, use complex computer algorithms to calculate the maximum that the institution could lose in a single day’s trading. These models seem to work well in normal conditions but not, alas, during financial crises, which is arguably when it is most necessary to know how much value is at risk.

Mainly reared for wool production. They require cooler environment to have better wool growth. The rearing is restricted to hilly regions where the weather is cool which helps in better wool production.

• Qualitative
• Quantitative

Quality-Length of the wool, texture of wool, shininess/ brightness of wool.

During marketing, classified into 3 categories:

Grade I → Rs. 1000/ kg

Grade II → Rs. 600-800

Grade III → Rs. 400-600

LICENSE, REGISTRATION & INSURANCE FOR DOGS

To keep dog, license from local Municipality or Panchayat. Only when certified for vaccination against rabies is shown, license is given to rear dog.

Registeration of Pups

Pups from the registered parent should be registered as soon as it is born.

Pups should be registered with Kennel club. Registered usually where parents are registered. Immediately after the birth, pup should be registered.

Dog Show

Most registered Kennel clubs conduct Dog show. They allow the dog to participate only if it is registered with the club. Usually Kennel club avoid prizes to animal. To give prize, they should have the following classification :