eChapters

Nitrates and nitrites are known to have injurious effects on animals when excess amounts are ingested. Nitrites are much more toxic but nitrates are reduced to nitrites by bacterial action in the digestive tract

Nitrites (NO₂) are rarely found in plants  in appreciable amounts except under very unusual conditions such as severe disease infections; therefore, the results are usually reported in terms of nitrates (NO₃). The usual method is to determine the total nitrate and nitrite in terms of nitrate. However, the method can also be used to determine the nitrite content of a feed separately if a nitrite problem is suspected.

Two procedure are described for the determination of free gosssypol . The first for normal meals and the second for meals which have been chemically treated and so contain dianilinogossypol.

Reagents
 
    1.    Aqueous acetone:  7 parts acetone, 3 parts distilled water (V/V).
    2.    Aqueous acetone-aniline solution: To 700 ml acetone and 300 ml distilled water, add 0.5 ml redistilled aniline. Prepare solution daily.
    3.    Aqueous isopropyl alcohol solution: 8 parts isopropyl alcohol. 2 parts distilled water (V/V).
    4.    Aniline Distill reagent grade aniline over a small quantity of zinc, dust discarding the first and last 10 percent of the distillate. Store refrigerated in a brown glass stoppered bottle. Solution is stable for several months.

Activation : The process of causing a substance to become artificially radioactive by subjecting it to bombardment by neutrons or other particles.

Activation Analysis : Any analytical procedure permitting the detection and measurement of trace quantities of elements following their exposure to a flux of neutrons.

Activity :  The strength of a radioactive source. In absolute units, it relates to the number of radioactive atoms decaying per unit of time. It is also a synonym for radioactivity. Absolute activity is usually expressed in curies or millicuries.

Alpha Particle : A particle which is identical to the helium nucleus, consisting of two protons and two neutrons. It carries a positive charge of 2.

Alpha Rays: A stream of helium nuclei. The helium nucleus has a mass number of 4 and an atomic number of 2. If consists of two protons and two neutrons.

Ionization Chambers for Gases

Generally these type of instruments are known as dose-rate meter (e.g., the so called “cutie-pie”). Radiation intensity (i.e., a constant stream of particles or photons) gives rise to a continuous series of pulses, and if these are allowed to merge, they form a weak electric current, which may be amplified and registered by an electronic circuit. The final scale reading will then be a measure of the energy dissipated in the following chamber per unit of time by the ionizing particles or photons. In gas-flow counters, the radioactive samples are placed inside the detector which will be transfused by a gas at atmospheric pressure. This instrument counts particles of low energy, such as the b particles from 14C, effectively (Window-less counting).

We may study the nutrient metabolism in a better way as explained in the following summarized form in Table 1.

Studies on Volatile Fatty Acids Production and Kinetics of VFA Pool as Determined by Using “Isotope Dilution Technique”

(Method of Krishna and Ekern, 1974a,b and c)
Details of Experiment
Materials
    (i)    Animals : Two fistulated sheep of Dala Texel breed weighing about 72 and 76 kg are selected. 
    (ii)    Experimental Diet :
        Basal Diet : Two experimental sheep are maintained on 1.1 kg early cut timothy hay per day. The ration is divided in four equal parts and offered four times a day during the preliminary period of about twenty days. One day before infusion of labelled VFA, the sheep are fed regularly at one hour interval (1.1 kg hay divided in equal parts) to help in increasing the mixing rate of rumen ingesta. The animals are given water adlibitum but they are not allowed to drink water during infusion. Digestible energy and metabolizable energy (ME) of the basal diet is determined by conducting a separate metabolism trial on the same sheep.

Extent of Passage
(Method of Krishna and Ekern, 1974D)
Definition of Terms
The term  extent of passage is used to describe how far a given ingested material travelled  or was allowed to pass. The point at which  undigested residue of the food comes to or has passed through the alimentary tract may be found by the use of markers without disturbing the subject. In some conditions of digestive system malfunction or disorder , the food residue may be blocked at point along with digestive tract.

Transit Time
This is the time it takes the digesta of a meal to pass through the alimentary tract or segments of it. This time also represents the  retention time in the tract or the particular segment. A simple way of calculating it is by recording the time of first or last appearance of the marked residue of a meal. Other more useful methods for calculating retention time include “mean retention time” (Castle 1956a), “mean time” (Blaxter et al., 1956) and “turnover time” (Hungate, 1968).

TVFA Estimation in Blood
(Method of Friedemann, 1938 modified by Mc Anally, 1944)

A method for the determination of volatile fatty acids in various biological materials has been described by Friedemann (1938) which is said to be applicable to blood, then the blood is added to dilute sulphuric acid, then sodium tungstate solution and solid magnesium sulphate are added and the whole is steam distilled.

Reagents
    1.    Saturated magnesium sulphate solution to which has been added 2.5 per cent by volume of concentrated sulphuric acid.

Introduction

Casein has been widely used as a substrate for measuring the trypsin inhibitor activity of natural trypsin inhibitors such as those which occur in Soybeans and other legume. The most common method employing casein is the one originally described by Kunitz (1947) which involves the spectrophotometric determination of the breakdown products produced by a given concentration of trypsin in the presence and absence of the inhibitor. On the other hand, the tryptic hydrolysis of a synthetic substrate such as benzoyl-DL-arginine-p-nitroanilide (BAPA), first introduced by Erlanger et al. (1961), does follow a zero order reaction, and one obtains, within broad limits, a linear relationship between the quantity of p-nitroaniline released and the concentration of the active enzyme. Although BAPA has been used for assaying the antitryptic activity of soybean fractions (Sambeth et al., 1967).

(Colorimetric Method of Barker and Summerson, 1941)
I. Method for Blood / Plasma
Principle

In this method, glucose and other interfering material of the protein free blood filtrate is removed by the Van Slyke-Salkowski method of treatment with copper sulphate and calcium hydroxide. A part of the resulting solution is heated with concentrated sulphuric acid to convert lactic acid to acetaldehyde, which is then determined by reaction with p-hydroxydiphenyl in the presence of copper ions.

Answer the followings.
    Q.1.    Which factors govern the efficacy of intramammary drug in mastitis therapy?
    Ans.    The important factors are spectrum of bacteria controlled, diffusibility through mammary tissue and cost of the drug.

    Q.2.    What should be line of emergency treatment when type of infection is not known?
    Ans.    Frequent removal of milk manually or by using oxytocin, broad spectrum antimicrobials parentrally as well as via intramammary route and non steroidal anti-inflammatory drug.

    Q.3.    Why therapy of mastitis often fails to respond in lactating cows in spite of intensive veterinary service?
    Ans.    The main reasons are as follows:
    a)    Ineffective drug diffusion
    b)    Inactivation of antibiotics by milk and tissue priteins
    c)    Formation of micro-abscesses which make inaccessible of drug to pathogens
    d)    Inefficient killing of bacteria and intracellular survival of bacteria

Fill in the blanks

    Q1.    …………… is a contagious pathogen in bovine mastitis
    Ans.    Streptococcus agalactiae

    Q.2.    Streptococcus agalactiae causes ……… (acute/chronic) mastitis if control procedure of mastitis is not used.
    Ans.    Chronic

Answer the following
    Q.1.    Mention the strategy to reduce the prevalence of Streptococcus agalactiae mastitis at herd level.
    Ans.    Good hygienic measures, proper disinfection and efficient treatment of clinical cases.

    Q.2.    What is the relation between SCC and Streptococcus agalactiae infection?
    Ans.    The possibility of Streptococcus agalactiae infection is high with high bulk milk somatic cell count.

    Q.3.    What are the common sources of Streptococcus agalactiae infection in a dairy herd?

Fill in the blanks

    Q.1.    ---------- and -------------- are the synonyms of mastitis of sheep.
    Ans.    Bluebag, garget
    Q.2.    In gangrenous mastitis, besides Staphylococcus aureus, ----------- organism may also be involved.
    Ans.    Clostridium perfringens A
Answer the followings

    Q.1.    What are the characteristic symptoms of mastitis in sheep?
    Ans.    The important symptoms include severe necrotizing inflammation of mammary gland, severe systemic reaction and reduced milk production
    Q.2.    Classify the sheep mastitis based on etiology and clinical symptoms.
    Ans.    a) Gangrenous mastitis by Staphylococcus aurcus mastitis b) Herd mastitis by Pasteurella hemolytica
    Q.3.    When the risk of mastitis in sheep is high?
    Ans.    During summer month between parturition and weaning

    Q.1.    What is the cause of mastitis in non-lactating mare?
    Ans.    Mastitis may develop in a non-lactating mare due to abnormally high estrogen levels in her system. This excess female hormone can result from a pituitary gland tumor or consumption of high estrogen containing hay. High estrogen concentrations can cause mammary gland enlargement and trigger the production and secretion of milk, even though the mare is not pregnant and has not foaled. If the non-lactating mare begins to secrete milk due to excess estrogen, she may become a candidate for mastitis.

    Q.2.    Why equine mammary glands are less prone to mastitis than bovines?
    Ans.    The possible reasons are as follows:
    a)    Mammary glands are relatively smaller in size
    b)    Concealed position of the glands
    c)    Small milk capacity of the glands
    d)    Greater resistance to infection in equine mammary glands because of differences in endocrine environment and local immunity

    Q.1.    What are the common infectious diseases affect teat skin of mammary gland?
    Ans.    The common infectious diseases that affect teat skin of mammary gland are cowpox, buffalopox, pseudocowpox, bovine ulcerative mammillitis, black pox, foot and mouth disease, vesicular exanthema, swine vesicular diseases, contagious exanthyma, Staphylococcus sp., Pasteurella sp etc.

    Q.2.    What is thelitis?
    Ans.    Thelitis is the inflammation of tissues of teat wall caused by traumatic injury to the teat cistern. It is generally manifested with gangrenous mastitis.

    Q.3.    What is the main etiology of gangrenous mastitis?
    Ans.    Staphylococcus aureus

    Q.4.    What are the causes of coming blood in milk and mentions its treatment?

Materials Required 

Fruits/vegetables, hand refractometers, fruit pressure tester, weighing balance, scale, chemicals and glassware required for analysis.

Procedure

    1.    Go to the orchard or vegetable field and collect samples of fruits and vegetables
    2.    Apply the specific maturity indices to the samples

Materials Required

Fruits/ vegetables, weighing balance, plastic crates, CFB boxes or other packaging material fruit pressure tester, hand refractometer, markers etc.

Procedure

    1.    Harvest the fruits
    2.    Subject them to various pre-treatments (as discussed in previous exercise).
    3.    Record their initial parameters i.e. weight, firmness, surface colour, TSS, rotting etc. 
    4.    Place properly packed commodity in storage alongwith a check under ambient conditions
    5.    Record the observations regarding changes in quality during storage
 

Materials Required

Fruit, pulper, fruit mill, hydraulic press, screw type juice extractor, stainless steel pans, chemical preservatives, glass bottles, closures, crown corks, crown corking machine, plastic barrels.

Procedure

    1.    Select and weigh sound fruits/vegetables for extraction of juice/pulp.    
    2.    Wash and peel the fruits as required.
    3.    Extract the juice by hot or cold process.
    4.    Pasteurize the juice/pulp to inactivate the enzymes and partially kill microorganisms.
    5.    Preserve the juice/pulp by heat processing after hot filling and hermetic sealing or by addition of permissible quantities of suitable preservatives.
    6.    Label the bottles properly and keep them for storage

Materials Required

Fruits or fruit pulp, steel pans, weighing balance, refractometer, sugar, salt, spices, acetic acid, citric acid etc. 

Procedure

    1.    Extract fruit pulp as told in earlier exercises. 
    2.    Take a known quantity of freshly extracted or preserved pulp.
    3.    Weigh sugar (about half the weigh of pulp).
    4.    Heat pulp and sugar over a gas flame.
    5.    Add spices, chillies, salt, citric acid (if required) etc. and cook
    6.    Record the TSS to judge the end point. It should be more than 50o Brix.
    7.    Add measured quantities of acetic acid and sodium benzoate at the end.
    8.    Cool and pack in pre-washed and sterilized glass jars
    9.    Label and store.

Materials Required

Fruits or vegetables, stainless steel knife, steel pans, Can Body Reformer, Flanger, Double seamer, weighing balance, Refractometer / Salometer, sugar or salt etc. 

Procedure

    1.    Wash the selected fruit or vegetable (as the case may be) 
    2.    Cut into pieces or slices (the size and shape may depend on the fruit/ vegetable but it should not be too large or too small)
    3.    Blanch the material in boiling water for 1-2 min for effective inactivation of enzymes. 
    4.    Prepare the sugar syrup of strength 40o Brix or brine of 1-3 % strength.
    5.    Prepare the can body using a can body reformer and flange the ends in a flanger.
    6.    Seal the base plate using first and second roller operations of the double seamer. 
    7.    Transfer the prepared fruits / vegetables into the cans. 
    8.    Pour in hot syrup or brine into the cans.
    9.    Place the lid over the top of filled can. (follow the first roller operation of the double seamer, if the cans are OTS cans)
    10.    Exhaust the cans in boiling water for about 10 min. to exclude any dissolved air out of the tissues or syrup/brine.
    11.    When the temperature of the centre of can reaches 79-80 ºC for fruits or 82ºC for vegetables, remove the cans from the exhaust box and seal them properly (follow the second roller operation of the double seamer, if the cans are OTS can).
    12.    Process the sealed cans at 100ºC and 115-121ºC for fruits and vegetables respectively for 15-30 min in case of fruits and 25-45 min. in case of vegetables.
    13.    Cool the cans immediately in cold water and air dry them at room temperature.
    14.    Label and store in a cool dry place.

Materials Required

Canned product, weighing balance, can cutter, vacuum/pressure guage, seam checking guage, Refractometer / Salometer etc. 

Procedure

    1.    Take a canned product say canned peach.
    2.    Record the weight of the can (A)
    3.    Record the information marked on the label i.e net  weight, date of packing, etc
    4.    Observe the can externally for the concavity of the lid.
    5.    Record the vacuum of the can by using a vacuum guage
    6.    Cut the lid of the can with a can cutter
    7.    Record the head space
    8.    Put all the contents over a sieve and let the liquid drain
    9.    Record the weight of empty can (B) to calculate net contents
    10.    Record the drained weight of the pieces.
    11.    Record whether the liquid is syrup or brine
    12.    Record the TSS of the salometer reading of the liquid (as the case may be)

Materials Required

Cut flowers, 1000 ml conical flasks, scissor, tape, distilled water, chemicals, weighing balance, volumetric flasks, labels, thermometer (max. & min. type) and hygrometer. 

Procedure

    1.    Collect fresh spikes of flowers
    2.    Cut at the base keeping about 25-60 cm of the cut flowers stick (uniformly for each flower).
    3.    Prepare Pulsing/ Vase solutions.
    4.    Put the prepared flower spikes in labeled conical flasks containing different Pulsing/ Vase solutions (for a known period of time).
    5.    Record the weight of the spikes and other observations daily.
    6.    Record the temperature and relative humidity of the storage chamber daily.
    7.    Analyse the recorded data and draw conclusion about the best vase solutions for the selected cut flower.

Materials Required

Fruit, vegetable or product sample, hot air oven, weighing balance, crucibles, petri plates, lead pencil / marker etc.

Procedure

    1.    Take crucibles  or petri plates and record the tare weight of crucibles / petri plates (C) and mark them
    2.    Cut the sample into small and thin pieces (if solid)
    3.    Put the sample into the crucibles / petri plates in three replications
    4.    Record the weight of crucibles + sample (C + S)
    5.    Put in hot air oven for drying at 70 + 2ºC.
    6.    Record the loss in weight after every 1-2 hrs till it becomes constant
    7.    Calculate the moisture and total solids. 

Materials Required

Sample, hot air oven, weighing balance, crucibles, muffle furnace, lead pencil / marker etc.

Procedure

    1.    After following the procedure for the determination of moisture (in silica crucibles as given in previous chapter) the same sample can be used for the determination of ash. 
    2.    Heat the dried sample in the silica crucibles over a heater/hot plate/burner to burn the carbon.
    3.    When it becomes completely black and no more smoke comes out of the sample, shift them to muffle furnace.
    4.    After heating the samples in muffle furnace, all the marks of pen / pencil/ marker are lost, so one would not be able to identify the samples and error of weights may occur. Therefore, it is advised to place the samples in the furnace in one or two rows and draw the diagram on a piece of paper for identification purpose.

Materials Required

Fruit/ sample, refractometers, thermometers, standard solutions of known TSS, temperature correction Tables.

Procedure

    1.    Calibrate refractometers of all ranges to be used.
    2.    The TSS of the juices/pulps/fresh fruits can be measured with the one having lowest range i.e. 0-32º Brix while, for measuring the TSS of concentrates, syrups, squashes, jams, jellies etc. refractometers of the higher ranges are made use of.
    3.    Put a drop of material on to the glass surface of refrectrometer’s prism, cover gently with the lid and observe the TSS against light.
    4.    Measure the temperature of the liquid which is being evaluated and apply temperature corrections (Annexure III).
    5.    Record the corrected value as the TSS of the sample.

Materials Required

Sample, reagents, beakers, conical flasks, pestle and mortar, funnels, volumetric flasks, pipettes, weighing balance, spectrophotometer/colorimeter.

Reagents

    1.    2% Metaphosphoric Acid Solution: Weigh 20 g of HPO₃ and dilute to 1 litre in water or 4% oxalic acid.
    2.    Dye Solution: Dissolve 100 mg of 2, 6 Dichlorophenol-indophenol dye (Phenolindo-2,6- dichlorophenol sodium salt, AR, C₁₂H₆Cl₂NNaO₂ 2-3H₂O) and 84 mg of sodium bicarbonate (sodium hydrogen carbonate AR, NaHCO₃) in hot (85-95ºC) distilled water. Cool and make up the vol 100 ml. Filter and dilute 25 ml of the dye to 500 ml with distilled water. 
    3.    Standard Ascorbic Acid: Weigh 50 mg of ascorbic acid (AR) and make up to 50 ml with 2% metaphosphoric acid or 4% Oxalic acid solution (1000 ppm i.e. 1000 µg/ml). Dilute 4 ml of this solution to 100 ml with 2% HPO₃ or 4% oxalic acid. (1ml = 40 µg ascorbic acid).

Materials Required

Specific gravity bottles, weighing balance, sample, pipettes, thermometer, distilled water 

Procedure

    1.    Take the sample and distilled water record their temperatures (they should be same for correct estimation). If the temperatures are not same bring both to 20ºC or the room temperature.
    2.    Clean and dry the specific gravity bottles properly before use.
    3.    Record the tare weight of the specific gravity bottle (T) alongwith the stopper.
    4.    Carefully fill the specific gravity bottle with distilled water up to the brim and then place the stopper tightly so as to exclude extra water. 
    5.    Dry the exteriors of the bottle with a tissue paper
    6.    Record the weight of (bottle + water) say A
    7.    Similarly, fill the same specific gravity bottle with test sample and record the weight (bottle + sample) say B
    8.    Calculate the specific gravity of solution using the following formula

Materials Required

Spectrophotometer, ethyl alcohol, beakers, pestle and mortar etc. 

Procedure

    1.     Preparation of sample extracts
        a.    Liquids : Centrifuge the juice at 5000 rpm for 4-5 min., and collect the supernatant. For liquid samples prepare a 40 % solution in ethyl alcohol. To 30 ml of the sample add 45 ml of ethyl alcohol and mix thoroughly. Filter through Watman 
No. 1 paper.

        b.    Semi-solid samples: To 15 g of blended sample add 15 ml of distilled water and 45 ml of ethyl alcohol. Mix thoroughly and filter through Watman No. 1 paper.
        c.    Dried products: Extract known quantity of sample (4-6 g) with 100 ml of 60% ethyl alcohol for 12 hrs and filter through Watman No. 1 filter paper.
    2.     Measure the absorbance of the samples at 440 nm for non-enzymatic browning and 400 or 420 nm for measurement of colour.
    3.     Prepare 60% ethyl alcohol solution as blank. Add 30 ml of distilled water to 45 ml of absolute alcohol and use as blank.

Materials Required

Sample, Liebig condenser, digestion flasks (700-750 ml cap), conical flasks, filtering cloth (thick linen cloth for clear filtration) reagents, muffle furnace, crucibles.

Reagents

    1.    0.255 N Sulphuric acid solution : Dilute 2.5 g (1.36 ml) of concentrated H₂SO₄ to 200 ml. Ensure correct concentration by titration.
    2.    0.313 N Sodium hydroxide solution: 2.504 g NaOH / 200 ml water nearly free from sodium carbonate. Ensure correct concentration by titration.
    3.    Asbestos Chips: gooch grade, medium fibre, acid wash and ignited
    4.    10% Potassium sulphate (K₂SO₄) solution: Dissolve 10 g of Potassium Sulphate (AR) in water and make up to 100 ml.

Materials Required

Soxhlet apparatus, petroleum ether, water bath, sample, grinder, weighing balance, filter paper, threads.

Procedure

    1.    Take the given sample and weigh it (A)
    2.    Dry the samples in an oven till there is no more weight loss and weight it (B)
    3.    Grind the sample into powder.
    4.    Place a weighed quantity of sample in filter paper, wrap the paper and tie it tightly with a thread so that no powder comes out of the paper during extraction. 
    5.    Pour approximately 100 ml of anhydrous ether into the bottom flask. Turn the heaters on. Set the temperature at about 40ºC.  
    6.    The ether will evaporate and circulate through the sample and again will come back to the flask. 
    7.    Repeat the process for about 16 – 18 hrs for complete extraction. 
    8.    Remove the sample and evaporate most of the ether from the mixture of crude fat and ether obtained during extraction.
    9.    Evaporate excess of ether from the crude fat sample by evaporation on a water bath. Evaporate till it is free from the fumes of ether. Record the weight (C).

Materials Required

Sensory scoring card, products, pen/ pencil for coding, plain water

Procedure

    1.    Assign codes to the samples i.e. a, b, c, …… or 1, 2, 3, ….. etc.
    2.    Keep the coded samples randomly (eg. c, f, a, b, d, e) in a row for evaluation
    3.    Start the evaluation of samples for various sensory attributes one by one. 
    4.    Evaluator should rinse his/ her mouth after each sample so as to avoid overlapping of taste, flavour etc.
    5.    Assign marks from 1-9 based on pleasurable and un pleasurable experiences.
    6.    Compute the means for a particular attribute on the basis of the evaluation of different evaluators (replications).
    7.    Find out the best sample on the basis of highest average scores obtained.

Materials Required

Sensory scoring card, products, pen/pencil for coding, plain water.

Procedure

    1.    Take the given two samples
    2.    Make them three by duplicating/repeating either one of the two
    3.    Assign codes to the samples eg. AAB, ABA, ABB,  BAB etc.
    4.    Ask the evaluators to evaluate the samples for any differences between the 3 on the given Specimen Evaluation Card.
    5.    Find out how many of the evaluators have chosen the odd sample correctly (say it is 16 out of 20 evaluators).
    6.    Now find out the degree of difference in the samples indicated by the evaluators who correctly found the odd sample (Say it is much as per 3, moderate as per 8 and slight as per 5 out of the 16 correct evaluators.).
    7.    Find out how many of the correct evaluators preferred A sample and how many the B sample (say 11 preferred A sample and 5 B sample).
    8.    After going through Annexure II you may conclude that the two samples differed significantly from each other as 16 out of 20 evaluators found the odd sample correct. But, the preference was not significant as it should have been at least 13 out of 16 judges indicating same preference so as to make it significant. So, it may be concluded that the two samples differ significantly but both are acceptable.

Materials Required

Sensory scoring card, products, pen/ pencil for coding, plain water.

Procedure

    1.    Assign codes to the samples i.e. a, b, c, …… or 1, 2, 3, ….. etc.
    2.    Keep the coded samples randomly (eg. c, f, a, b, d, e) in a row for evaluation
    3.    Start the evaluation of samples one by one. 
    4.    Evaluator should rank the samples on the basis of intensity of a single sensory attribute according to his / her preference.
    5.    Compute the means on the basis of the evaluation of different evaluators (replications).
    6.    List the samples in increasing or decreasing order of preference.

Materials Required

Record book

Procedure

    1.    Follow the instructions given by the teacher and the factory representative.
    2.    Listen carefully to the explanations of the factory representative
    3.    Observe the functioning of various equipments
    4.    Ask queries wherever not understood properly.
    5.    Note down the observations in your record book  

Observations

    1.    Name and address of the factory or processing unit
    2.    Size of the Unit (Large, Small, Cottage, Home)
    3.    FPO Licence No.    
    4.    List of products manufactured    
    5.    Height of the processing plant    
    6.    Information regarding raw material store, finished product storage    
    7.    List of equipments installed in the factory
    8.    Water supply system     
    9.    Waste disposal system    
    10.    Hygiene in the surroundings    
    11.    Any other

Genetic architecture of a population is the result of prolonged natural selection. The populations which exist in diverse environments might have been strongly diversified genetically. So, it is necessary to understand the extent and rates of genetic divergence existing between the diversified forms or types. Genetic diversity must be sufficient enough to justify breeding effort. Genetic diversity may be available in improved germplasm or it may exist only in genetically inferior stocks. The availability of statistical tools to quantitatively measure the genetic divergence between two or more populations and the relative contribution of individual characters to the total divergence have permitted to trace the evolutionary patterns in some crops such as rice and tobacco and in choosing the parents for hybridization in crop plants (Rao, 1958; Blackith, 1960; Morishima and Oka, 1960; Murthy et al.,1962). Among the several statistical methods developed for measuring the divergence between population, multivariate analysis (D² statistic) developed by Mahalanobis in 1936, has been found to be potent tool.

Mahalanobis D² analysis is an effective tool in quantifying the degree of divergence at genetic level and provides a quantitative measures of the association between geographic and genetic diversity based on generalized distance.

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.

Koch‘s Postulates (Pathogenicity test)

Robert Koch developed the following postulates in 1883 on the basis of which an organism could be attributed to be the cause of a specific disease.

i)Recognition: The suspected organism must be found in every case of disease (Presence of organism in the diseased tissues).

ii)Isolation: The organism must be isolated in pure culture from every case of disease (Isolation in pure form).

iii)Inoculation: The isolated pure culture when introduced into the same species or variety of susceptible host must be capable of reproducing the original disease with its typical symptoms (Pathogenicity).

iv)Re-isolation: The organism must be re-isolated from the artificially infected host and its characteristics must be exactly like those observed in step 2 (This step was added by E.F. Smith).

If all the postulates are proved true, then the isolated pathogen is identified as the actual causal organism responsible for the disease.

Introduction

Chemicals that kill or retard fungi are fungicides. Most chemicals used to control plant diseases are ”protectants“ and must be applied before infection to protect the plant from invasion by a pathogen. Protectant fungicides are usually effective against a broad range of fungi and protect the plant against infection on the surfaces of the plant to which they are applied. Often, they require multiple applications during the growing season to maintain coverage as new growth emerges and weathering removes past coverage. A few chemicals are termed ”eradicants“ since they can eliminate an established infection. Some newer fungicides are systemic, meaning that they are absorbed by the foliage or roots and move within the plant to the site of infection. Systemic chemicals may be protectants, eradicants, or both systemic fungicides can be absorbed by the plant without harming it, and transported to other tissues where they are toxic to fungi. These compounds can control and eradicate established infections, but they are also vulnerable to fungi developing resistance, as they generally only target one step in a biosynthetic pathway to kill the fungus. To minimize the development of resistance by chemical overuse, fungicides are classified into groups based on their chemical activity.

Preparation of Bordeaux mixture

Materials: Iron, beaker, copper sulphate, lime, weighing balance, plastic container, wooden stick and muslin cloth.

Bordeaux mixture derives its name from the place of its discovery, Bordeaux, France.

Ingredients required for the preparation of 100 litre of 1 percent elicoid mixture are:

1.Copper sulphate (blue vitriol or bluestone) : 1 kg

2.Quick lime (unslaked) : 1 kg

3.Water 100 litres

Method

•Dissolve 1kg powdered copper sulphate crystals in 50 litres of water in a mudpot or plastic bucket.

•In another 50 litres of water dissolve 1kg of quicklime.

•Stir thoroughly with the help of wooden stick.

•Put a filter cloth on the mouth of 3^rd container.

•Pour the contents of both the containers in a 3^rd container simultaneously and stir it thoroughly.

•Make up the desire volume (ie. 100 litres).

•Test the mixture before use for the presence of free copper, which is harmful to the plant.

Introduction

Diseases are a common occurrence on plants, often having a significant economic impact on yield and quality, thus managing diseases is an essential component of production for most crops. These fungicides are used because of three main reasons:

a.To control a disease during the establishment and development of a crop.

b.To increase productivity of a crop and to reduce blemishes.

c.To improve the storage life and quality of harvested plants and produce.

Application Methods

To protect the crop from fungal pathogens, fungicides are applied in various formulations (as dust, granules, gas and most commonly, liquid). They are applied to:

1.Seed treatment

2.Soil treatment

3.Protection of standing crop

Poisoned food technique

The principle involved is to poison the medium with fungi toxicants and then allow a test fungus to grow on such medium. The test may be performed both on solid and liquid medium.

Procedure

On solid medium

•Prepare potato dextrose agar medium.

•Add required amount of fungicide and thoroughly mixed.

•The medium is poured into sterilized Petri-plates.

•A culture of test fungus is grown on PDA for a certain period (generally seven days) at the optimum temperature for growth.

•Small disc (7 mm) of fungus culture is cut with sterile cork borer and each Petri plates are centrally inoculated aseptically containing the medium with desired fungicidal concentration.

•Suitable checks are kept where the culture discs are grown under the same conditions on PDA without fungicide.

•The fungal colony diameter is measured at every 24 hr.

Year         Milestones

1951       :    First amino acid was sequenced by F. Sanger and H. Tuppy.

1953    :    J.D. Watson and F.H.C. Crick proposed that DNA is a double helix.

1958    :    Discovery of DNA polymerase by Kornberg’s group and DNA replication studies began.

1959    :    Isolation of RNA polymerase.

1970    :    Discovery of reverse transcriptase showed that RNA can act as the template for the transcription of DNA and opened the way for cloning using cDNA libraries.

1970    :    General method was developed by M. Mandel and A. Higa for introducing DNA into cells by calcium dependent method.

1970    :    G.H. Khorana synthesized an artificial gene from DNA nucleotides.

1971    :    Restriction endonucleases for mapping DNA was first used by K. Danna and D. Nathans.

1972    :    The recombinant DNA Era begins. J. Mertz and R.W. Davis used T4 ligase to join DNA molecules.

Food is basic to human being and agriculture is the catalyst between the two which is basically supported by natural resources. The human being is entirely dependant on food and food is fully dependent on agriculture and humanity. Eventhough food, agriculture and humanity have their own entity; they are inseparable for the survival and existence of each other. Hence, the three pillars of food, agriculture and humanity forming a triangle symbolize the roots of human civilization.

I find myself fortunate to have the opportunihj to live and experience the working life of the farmers and the farming in Kansas (USA), Nigeria (Africa), Saudi Arabia and Yemen (Middle East), Italy (Europe) and India, Myanmar and Thailand (Asia). It zvas fiirther strengthened by visits to farmers in another about 100 countries. Day after day and month after month and even years, it made me to realize that community culture, language and farming in each state, country and continent are different from each other but some things are common and that they are all doing farming, they are growing food and they all doing for the livelihood of their families, neighbors and communities near and far (Nath, 2011).

The foundation of agriculture is laid on available natural resources e.g. land, water, sunlight, air, plant, animal and human beings. The human beings, being the leader to manage the resources have contributed to redefine agricultural science and promote and develop technology. This has led to augment agriculture and its science and technology. At the end, the human efforts have brought about changes and improved agriculture production at the cost of excessive use of natural resources, knowing well that without natural resources, without good soil and water hardly anything will survive.

Presently world produces enough to feed the present human population, but it is not available to millions of people. It is not the lack of availability of food but is the unequal distribution and lack of entitlement which are causing hunger and malnutrition in millions of population.

Objective: To estimate total protein in the plant sample by Folin-Lowry's method.

Background information:

Protein assay can be done to determine the level of protein in solution by the Folin-Lowry biochemical method. The concentration of protein leads to change in color of the solution and based on different color in different protein concentration solution the change in color may be digitized by colorimeter or spectrophotometer. This technique was developed by Oliver H. Lowry in the year 1940s. The tyrosine and trytophan amino acid residues in a protein sample produces purple or blue color in the absorption maxima at 660 nm by using Folin-Ciocalteau (having phosphate and sodium tungstate molybdate) chemical reagent. The color intensity of protein sample depends on the concentration of above aromatic amino acids (tyrosine and tryptophan), hence every sample will vary for color. For protein estimation the BSA (Bovine serum albumin) is widely used as standard protein molecule due to its cost effectiveness, purity and easy availability. This method of protein estimation is very sensitive and can detect up to 10 μg/ml. This is a relative method of protein estimation and may be interfere by salts, Tris buffer, nonionic & cationic detergents, EDTA, lipids and carbohydrates. For reproducible assay the incubation time, pH (9-10.5) is critical in this method (Lowry, et al. 1951).

Objective: To extract and perform enzyme assay of polyphenol oxidase from plant.

Background information:

Polyphenol oxidase (PPO or monophenol monooxygenase or polyphenol oxidase I, chloroplastic) is a tetramer that contains four atoms of copper per molecule, and binding sites for two aromatic compounds and oxygen (www.worthington-biochem.com). The enzyme catalyses the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols (phenol molecules containing two hydroxyl substituents). It can also further catalyse the oxidation of o-diphenols to produce o-quinones.

PPO causes the rapid polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that cause fruit browning. The amino acid tyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone. Hence, PPOs are some time referred as tyrosinases (Mayer, 2006).

Jolley et al. (1974) referred to PPO as 4 electron-transferring phenol oxidase and oxygen. It is responsible for browning reactions throughout the phylogenetic scale.

Objective: To extract and perform enzyme assay of catalase from plant.

Background information

Catalase is a commonly distributed enzyme and present in approximately all organisms exposed to oxygen (such as bacteria, plants and animals). Catalysis of H₂O₂ into H₂O and O₂ is done by catalase enzyme (Goodsell, 2004).

Objective: To extract and perform enzyme assay of guaiacol peroxidase from plant.

Background information:

Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

Objective: To extract and perform enzyme assay of Superoxide dismutase from plant.

Background information:

Superoxide dismutase (SOD, EC 1.15.1.1) is the enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O₂^-) radical into either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, may cause cell damage (Hayyan, et al. 2016). Hydrogen peroxide is also responsible for cell damage and may be degraded by other enzymes like catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use a different mechanism to prevent damage from reactive O₂^-.

Objective: To check quality of genomic DNA on agarose gel electrophoresis.

Background information

Agarose gel electrophoresis is a common method of gel based electrophoresis technique used in biochemistry, molecular biology, genetics and clinical chemistry for separation of Protein and nucleic acids from a mixture of macromolecules. Agarose is a major component of agar that is used as a matrix in gel baseelectrophoresis. The nucleic acids (DNA and RNA) are separated based on length of fragments while, proteins are separated based on size and/or charge (Kryndushkin, et al. 2003). For separation of bio-molecules the electric field is applied that leads to movement of charged particles via agarose gel matrix and bio-molecul s are separated based on difference in size in the agarose gel matrix (Sambrook and Russel, 2001).

Gel casting with agarose is easy and has very few charged groups, hence found most suitable for nucleic acid separation in most of the biochemistry, molecular biology and biotechnology laboratories. Separated DNA/RNA ma be seen and recorded with stain under UV light. Further DNA fragments can be eluted from agarose gel with an ease for further down applications. The percentage of agarose gel used is in the range of 0.7 - 2. Lower percent of agarose gel is used for long DNA fragments while, higher percent of agarose gel is used for short DNA fragments with suitable running buffer.

Objective: To check the integrity of isolated RNA on agarose gel.

Background information:

The most common method used to assess the integrity of total RNA is to run RNA sample on agarose gel (denaturing) stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in nativegels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species.

Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a smear (having very low mw). Inclusion of RNA size markers on the gel will determine the band size or smears size and will also serve as a good control for ensuring proper gel run. Note: Poly selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).