eChapters

Nowadays, revolutionary changes in entire globe have led to the economic, cultural and scientific development in our society and have augmented considerable changes in our food habits and life-style. Continuously growing population had noticeably increased the pressure on land, water and energy resources. All these resources are scarce and going to be exhausted in coming decades at the present rate of consumption. Undoubtedly, production of food is more imperative than the production of energy or any other bioproducts. Moreover, with the world population rising to 9 Billion in 2050, with many inspiring to a western life style and diet, this challenge should be addressed urgently (Draismaa et al., 2013). The challenge to produce about 70% more food by 2050 for a growing and increasingly affluent world population has motivated several review papers addressing the different dimensions of the food system and food security (Verburg et al., 2013). Presently, human beings consume approximately 2.5 billion tons of dry weight of harvested crops which include approximately 2.3 billion tons of cereals (e.g., rice, wheat, and corn kernels) and grass from managed pastures (Smil, 2000).

Ever since the dawn of civilization, dependence on plants has been indispensable for human survival. Transformation from food gathering to settled agriculture necessitated domestication of plant species useful for mankind leading to co-evolution of plants and man in absolute harmony with nature. Natural disasters like famines, floods, epidemics have often led people to search new plant species for human consumption. Out of an estimated total of 80,000 plants of possible economic use, about 30,000 plants have been found to be edible in nature and approximately 7,000 plants have been cultivated by mankind at one time or the other, of which only 158 plants are widely used for food (Wilson, 1992). Among these, 30 crops provide 90% of the world’s food and only 10 crops supply 75% of the world’s food budget. Out of the above only three crops, rice, wheat and maize provide 60% of the world’s total food requirement (Paroda, 2000). This dangerously narrow level of food base may imperil the existence of mankind during a time of impending crisis in unforeseen times in the future.

Wild edible plants have played an important role in human life since time immemorial. People depend on the wild plant resources for various purposes like fire wood, timber, non-timber forest products, medicines and food (Pushpangadan, 1995). In many cases, rural communities depend on wild edible plants, to meet their food needs in periods of food shortage. Indian subcontinent is blessed with varied and diverse soil and climate conditions suitable for the growth of various plant species. It has been estimated that 9000 of the 15,000 higher plants occurring in India are commercially useful. Of these 7500 are medicinal, 3900 are edible, 700 are culturally important, 525 are used for fiber, 400 are fodder, 300 for pesticides and insecticides, 300 for gum, resin and dye and 100 for incense and perfume (Anonymous, 1992-1993).

The palmyrah palm exists both in the wild as well as in cultivation, ranging from sea level about 800 amsl. It is usually grown in seasonal tropical or subtropical climates on sandy soils. It is a very adaptable palm, however, growing well in dry areas and is quite drought resistant. It also grows in per-humid areas and survives water logging quite well. The worldwide distribution of natural and cultivated populations of the palmyrah shows that it occupies a band on either side of the equator and extends laterally over a considerable girth. The country wise distribution of the palms are Mauritania, Senegal, Mali, Gambia, Guinea-Bissau, Guinea, Ivory Coast, Upper Volta, Nigeria, Gaban, Congo, Sudan, Tanzania, Madagascar, Saudi Arabia, Iraq, Iran, Pakistan, Bangladesh, Sri Lanka, Myanmar (Lubeigt, 1977), Thailand, Cambodia, Malaysia and Indonesia (Kovoor, 1983). In India, the palms are wide spread in Kerala, Tamil Nadu, Karnataka, Andhra Pradesh, Maharashtra, Odisha, Madhya Pradesh, Bihar and West Bengal.  Isolated patches are also seen in Assam, Gujarat and Uttar Pradesh (Anonymous, 1948). The palmyrah is considered as a slow grower and the growth rate decreases in regions of higher altitude (Tjitrosoepomo and Pudjoarinto, 1982).

The Indian Himalayan region is a well known abode to numerous plants of medicinal significance. The Western Himalayan ranges are home to a diverse flora of variety of endemic medicinal plants making it a biodiversity-rich region (Samant et al., 1998).  The ancient system of traditional medicines has been using these endemic medicinal plants since time immemorial. Of late the resurgence of herbal system of medication has led to an unprecedented rise in extraction of such plants from the wild for medical preparations. Most of the times the plant collection is unscientific and unthoughtful of ecological status of the medicinal plant, especially herbs. As a result, unplanned collection and other human interferences have brought a threat to survival of several important medicinal plants of the IHR an  example being  Brahmakamal.

Tropical root and tuber crops are the third important group of food crops after cereals and pulses. Tuber crops hold a premier position among food crops in the tropical and subtropical regions of the world. These crops are rich in starch and calories and have great potential in meeting the food and energy needs.  More than 840 million people do not have enough food to meet their basic daily energy needs. Far more - an estimated 3 billion - suffer the insidious effects of micronutrient deficiencies leading to hidden hunger. Apart from its popular use as food, tubers also find an important space in cattle feed and agro industries sector.Tropical tuber crops possess high photosynthetic ability coupled with the capacity to yield under marginal lands, adverse biotic and abiotic stress etc (Ravindran et al., 2008).

Dragon fruit, commonly known as pitaya/pitahya (Hylocereus sp.) is a  climbing perennial cacti belonging to the Cactaceae family and the subfamily Cactoidea of the tribe Cactea (Raveh et al., 1993). It is a perennial climbing cactus plant native to tropical areas of North, Central and South America. Main countries growing dragon fruit are Vietnam, Colombia, Mexico, Costa Rica, Nicaragua, Thailand and Taiwan. Cultivation also occurs in Australia, Israel and Reunion Island. The European Union and Asia, especially China are the largest import markets of dragon fruits (Vinas et al., 2012).

Broad dhaniya (Eryngium foetidum L.: Umbelliferae - Apiaceae) is an underutilized leafy vegetable and aromatic plant used for seasoning food items and source of medicine. It is a shade loving plant of tropical regions.  It has got status of a promising cash crop with huge export potential particularly in United Kingdom, USA and Middle East markets. Its medicinal and seasoning properties are well known in various countries like Vietnam, India and in the Amazon region (De Gumao et al., 2002). Broad dhaniya is relatively unknown in many other parts of the world and is often mistaken and misnamed for its close relative Coriander (Coriandrum sativum L.).

Black caraway (Bunium persicum Boiss. Fedtsch ) commonly known as kala zeera is a low volume,  high value, under-exploited and  non-perishable crop that finds place  in cold arid  North -Western Himalayan regions of Indian sub-continent. Its fruits are expensive spice and are used in indigenous system of medicine for curing various ailments and disorders. The proper information on botanical identity of the species, its description and distribution, active ingredients, standardized agro-techniques, processing and value addition and marketing potentials, etc. are very scanty. An attempt has been made to gather information on various aspects of kala zeera to promote its cultivation in the cold arid regions of the Himalayan states and its role in improving the socio-economic conditions of the people by cultivating the cash crop.

Dyes are one of the most important by products of the plants, as they are related with cultural practices, rituals, arts and crafts, fabrics and to satisfy personal embodiment. However, dye yielding plants have not received significant attention (Grover and Patni, 2011). Dye is a substance that has affinity to the substrate to which it is being applied such as textile fibers, foodstuffs and powder. They are organic coloured substances which absorb light in the visible part of the spectrum and have the property of colouring the substrate they are applied to.
 
Colourants which are obtained from plants and natural sources are known as natural dyes. Natural dyes have been traditionally used in most of the countries. These dyes were obtained from roots, leaves and bark (Akhtar et al., 2012). Natural dyes find use in the colouring of textiles, drugs, cosmetics, etc. Owing to their non-toxic effects, they are also used for colouring various food products. In India, there are more than 450 plants that can yield dyes (Siva, 2007).

Kale (Brassica oleracea L. convar. acephala (DC.) Alef.) is a representative of cole group of vegetables and cool-season cooking green somewhat similar to collard and non heading cabbage that has gained recent widespread attention due to its health-promoting, sulfur-containing phytonutrients. Kale is grown for its large foliage which is mainly used as leafy vegetable. This leafy vegetable is helpful in reducing the chances of cancer if taken regularly. Among the vegetable crops, kale is the richest source of carotenoids which is the precursor of vitamin-A. Besides, it also contains good amount of different minerals essential for human diet. There are several varieties of kale, all of which differ in taste, texture and appearance.

Gingers  belong to Zingiberaceae family and are made up of about 1000 species in 47 genera. They have numerous members with aromatic oils that have made them popular for perfumes and ornamentals. Ornamental gingers, while related to edibles, are usually grown for their looks instead of their flavor. A very large and diverse group of gingers are valued both for their tropical foliage and for their large, colorful flower heads. From the startling red ginger to the subtle shell ginger, these attractive plants are easy to grow in home landscapes in tropical and semi-tropical areas. They are great for tub gardens They can be very rewarding to grow, and with some simple care will continue to produce beautiful leaves and flowers for many years which also have cut flower value.

The edible speciality mushrooms are mushrooms that are generally not grown and available commonly in the market. Any mushroom other than popularly grown white button mushroom (Agaricus bisporus) falls under the specialty category, and these mushrooms are generally not available over the counter or in departmental stores in India or other countries in the world. The mushrooms under this group include the different types of commonly grown Oyster mushrooms like grey, black, white, pink, yellow oyster mushrooms, King Oyster mushroom, Shiitake mushroom, Black Ear mushroom, Shimeji mushroom, Enokutake mushroom, Milky mushroom, the Portabella brown, Cremini mushroom , Straw mushrooms and others. This paper will describe in detail biology and cultivation of these mushrooms as grown under controlled environment conditions or under seasonal growing conditions, where ever possible.

Mizoram in North East India, is located between 21º58’ to 23º35’ N latitude and 92º15’ to 93º29’ E longitude, surrounded by Tripura, Assam and Manipur in North frontier regions; Bangladesh in west; and Mayanmar in east and south. The undulated topography of Mizoram, known as Lushai hills during British period, has varied altitudes ranging from 21 to 2157 m above mean sea level, annual rainfall of 2000-3200 mm, and mean monthly temperature of summer (monsoon) ranges from 14.6ºC to 29.6ºC. Besides having good rainfall; the vegetable production, including leaf vegetables, during winter period is limited by acute soil moisture stress caused by high rate of evapo-transpiration, almost no rain from November-March, non-availability of irrigation water and low water holding capacity of the sloppy land (Singh et al., 2011; Singh et al., 2013b).

Introduction and History

Leguminosae/Fabaceae is a superfamily of dicotyledonous plants, which in species diversity is the third largest family of plants accounting for 5% of Earth’s plant biodiversity and make up for more than at least 1% of the total Earth plant biomass. Among these, legume grains and pulses having more than 20 to 30% lysine protein content are cultivated in 145 million ha of land in developing countries. This family is also characterized by presence of specialized tumor like structure formation (nodules) on roots (occasionally on stem as in Sesbania rostrata, Aeschynomene) that is inhabited by microsymbiont gram-negative bacteria (Rhizobium), engaged in fixing atmospheric nitrogen or symbiotic biological nitrogen fixation.

Introduction

Rhizobia and Frankia are soil bacteria that exhibit symbiosis with their particular hosts owing to their ability to understand the intricate signals excreted by the host roots. Rhizobia exhibit symbiosis with legumes and one species member of Ulmaceae family Parasponia andersonii while actinomycetes Frankia forms the group of nitrogen-fixing bacteria that live in symbiosis with a large spectrum of dicot plants distributed among eight families consisting of over 24 genera and around 200 species of angiosperms (Benson and Silvester, 1993) and are collectively responsible for 15% of the biologically fixed nitrogen in the world. This Frankia-woody tree or dicotyledonous plant interaction results in evolution of the actinorhizal plants and the symbiotic interaction is described between Frankia (actino) and plant roots (rhiza).

Introduction

Nitrogen-fixing cyanobacteria are the group of most primitive prokaryotes that have been observed in proterozoic era. Many Proterozoic oil deposits are attributed to the activity of Cyanobacteria, such as Gloeocapsomorpha. Small concentrically layered structures called pisolites are also the result of fossilized bacteria.

Introduction

Diazotrophs are phylogenetically diverse microbes spanning over various classes varying vastly on basis of physiological properties (Young, 1992), include various phototrophic microorganisms aerobic phototrophic Cyanobacteria (Vaishampayan et al, 2001), anaerobic purple-sulfur phototrophs like Chromatium, and green-sulfur phototrophs, e.g. Chlorobium, Rhodopseudomonas; chemolithotrophs Alcaligenes, Thiobacillus, Methanosarcina, Methanobacterium, Desulfovibrio or Azospirillum lipoferum and heterotrophic bacterial anaerobes (Clostridium), microaerophiles (Herbaspirillum), and aerobes (Azotobacter). Free-living aerobic nitrogen fixing bacterial genera found in soil include obligate aerobes as Azotobacter, Beijerinckia, Derxia, Achromobacter, Mycobacterium, Arthrobacter and Bacillus but the majority are microaerophilic (e.g. Azospirillum, Herbaspirillum) or facultative and obligate anaerobes (e.g. Klebsiella, Aerobacter, Desulfovibrios, Rhodospirillum, Clostridium, Erwinia).

Introduction

Increased population pressure, reduced length of fallow, deforestation and improper agricultural practices have led to widespread soil degradation in many parts of the developing world which has culminated in appearance of determinative manifestations of environmental damage in form of inadequate replenishment of soil nutrients and organic matter. Phosphorus is second major plant growth limiting nutrient and despite being in abundance in soil (organic and inorganic form), P deficiency is becoming critical in many soils throughout world as these soils have free available P concentration (generally) not higher than 10mM even at most soluble slight acidic pH (Arnou, 1953). Moreover, consistent P deficiency threatens to disturb the use efficiency of applying other nutrients. Hence to preserve the sustainability of agriculture it is foremost to rebuild soil fertility and maintain/improve the soil biodiversity to reap hiked current levels of productivity and farm income.

Introduction

Lithosphere, including the earth’s crust, is helpful in providing the base or medium for sustenance of most life processes and forms, interwoven in fine network interdependencies within themselves and the environment surrounding them i.e. ecosystems. Terrestrial ecosystem contains an important dynamic living matrix termed soil, which is critical for agricultural production and food security. Soil forms the medium for growth and maintenance of a variety of plant species while the plant roots in the soil represent a four dimensional region in space and time of profuse activity relative to the bulk soil, revolving around pH, nutrient, redox potential, and exudate gradients changing as distance from the root increases (Marschner, 1995). The soil region adhering the plant roots is being strongly influenced by the presence of these roots and is characterized by high rates of microbial population and activity and referred as rhizosphere (the soil adhering to plant roots).

Introduction

Vesicular arbuscular mycorrhizae are fungi that exhibit symbiosis with roots of higher plants. The term vesicular arbuscular is derived from characteristic structure i.e. ‘branched hyphae or arbuscles’ that are formed with in the root cortical cells and vesicles or ‘swollen lipid storing organs’ that occur with in or between them.

Introduction

India being a developing nation is trying to reach a balance in ever increasing population with marginal increase in crop production. There occurred a bigbang in crop production during the green revolution era however this also resulted in creation of infertile, barren lands with low chemical nutrient input responsive soils. Soil fertility is the major constraint among resource poor Indian farmers as lesser benefits are harnessed by use of newer agricultural protocols/tools/products viz improved varieties, productive technology from less fertile soils. There is dire need for looking for the alternative technologies of food or crop production to satiate the hunger of burgeoning Indian population. Today there is enormous emphasis on the conservation facets of soil fertility and crop productivity which are the instrumental philosophies in sustainable agriculture. Biofertilizers are the cost effective, ecofriendly and renewable resource of plant nutrients that could well supplement the use of already functional and popular chemical fertilizers for obtaining increase in crop yield without over hazardous impacts on various facets of soil chemistry, fertility and microbial diversity to attain sustainable agricultural system.

Introduction

Introduction of improved high yielding varieties of crop plants, use of modern agricultural practices coupled with essential control of pests of food and fiber crops have significantly contributed towards increasing the food production in green revolution era in India. Pesticide application forms the foremost limb in hiked agricultural food production and management practices. The production and post production management of crop yield requires increased use of pesticides.

Introduction

Soil forms the base of the lithosphere on earth and is a fundamental and irreplaceable versatile three dimensional dynamic system that supports majority of terrestrial plant and animal life on earth. This soil system contains mineral (sand, slit and clay forming basic soil structure) and organic matter as well as marginal air and water contents. The soil organic matter content of the soil system constitutes every fraction of matter in various stages of decomposition that once was associated with the body, body parts, exudates, tissues and cells of living organisms including plants, animals and microorganisms. It is the product of continuous cycling of the compounds synthesized by autotrophic plants among the three kingdoms viz microbes, plants and animals. The decomposition of the organic residues in soil is performed primarily by the resident soil microflora termed as microbial decomposers; though initially the larger and complex organic material is broken down, devoured upon, chewed, regurgitated and transformed into smaller bits by larger soil faunal forms (earthworms, beetles, springtails, and collembola, the macro (large) to mesofauna) on which soil microbial forms could act upon.

Introduction

Bioremediation can be defined as elimination, attenuation or transformation of polluting or contaminating substances by the use of biological processes, to minimize the risk to human health and the environment. Bioremediation of contaminated soil and water uses microbes (e.g. bacteria and fungi) to convert pollutants to harmless or more environmentally acceptable products and minimizes the risk to human health in a natural or managed process.

Cell culture has become one of the major tools used in the life sciences today. Viruses replicate only within the living cells. With the advent of cell culture technique, it has become easier to grow the viruses under in vitro condition (cell culture). Viruses are usually propagated in cell culture, embryonated hen’s egg, laboratory animals or natural hosts. In 1949, Enders, Weller and Robbins first reported that polio virus could be grown in cultured non-neural cells with the production of recognizable cytopathic changes. Although the cell culture technique has been evolved long back, it is only since the advent of antibiotics that cell culture became a matter of simple routine technique. Aseptic precautions are still essential, but the problems of contamination with bacteria, mycoplasma, fungi and yeasts can be checked by antibiotics and anti-fungal agents. The term in vitro literally means ‘in glass’ although today most cell culture is performed in or on plastic.

The cell is the unit of structure of all plants and animals. With increasing complexity of organization, masses of different cell types tend to become localized and to form recognizable patterns. An aggregation of cells forming a definite pattern is called tissue. A still higher level of organization, tissues aggregate to form organs. The animal cell is a highly organized structure. Plant cell is different from animal cell  by having a cellulose cell wall.  There are a number of well defined structures viz., nucleus, mitochondria, lysosomes, Golgi apparatus etc in the cell. Animal cells also have centrioles whereas plant cells contain plastids such as chloroplasts. The remainder of the cell is the cytoplasm.

The best environment for growing cells is similar to  the conditions what they experience in vivo. A number of various environmental factors affecting the tissues are :
 
    (1)    Temperature : In living matter, complicated chemical reactions are happened at comparatively low temperature.  In most of the mammalian and avian tissues the temperature  is 37^ºC to 38.5^ºC. If the temperature is enhanced to 45^ºC, cells are destroyed within  an hour but survive 12-24 hours at 42^ºC. It is applicable to most of the fibroblasts and epithelial  types of cells arising  from  mammalian tissues. Some of the mammalian cells ( human skin epithelial cells)  and amphibian cells  prefer lower temperature but fish tissue will not survive beyond 20^ºC.

Types of cells : Animal cells are usually defined by the tissue of their origin. On the basis of morphology, they can be divided into 5 types.

    1.    Epithelial cells : Epithelial tissues consist of a layer of cells which cover organs and line cavity. Example skin and the lining of alimentary canal. They grow well as single cell monolayer in culture.

    2.    Fibroblast cells : Connective tissue form the major structural component of animals. The tissue contains fibroblasts and most widely used cell in the laboratory. Fibroblasts are bound to the fibrous protein collagen in the connective tissue. They have excellent growth characteristics.

A finite growth capacity is a characteristic of all cells derived form the normal animal tissue. The cells pass through a series of age related changes before being incapable to divide further. The finite number of generations of growth is a characteristic of the cell type, age and species of origin and referred to as the “Hayflick limit”. The cells derived from embryonic tissue have a greater growth capacity than those derived from adult tissue.

A cell culture laboratory should allow the sterile handling of cultured cells with no or minimum level of contamination. The growth of bacteria and fungi is very high and no level of contamination can be allowed in animal cell culture. However, it is impractical to design a laboratory free from potentially contaminated microorganism. Further, it is possible to establish a laboratory with minimal risk of contamination. Several points should be taken into consideration while designing a new cell culture laboratory.

Once cells are obtained from a culture collection or isolated from animal tissue,, cells are put in the flask with growth medium. The cells are inoculated at a density of 104-105 cells/ml to get a concentration of 106 cells/ml or 105 cells/cm2 in a solid surface in 2-3 days because of nutrient limitation, accumulation of toxic metabolite or lacking of growth surface.

Cell culture medium : To grow cells in in vitro, environment should be as close as possible to in vivo condition. But a number of factors are important to grow cells in vitro namely substrate, temperature and medium. There is a number of ready made medium available commercially and made the cell culture work easier. Any successful medium consists of isotonic buffer solution containing inorganic salts, amino acids, vitamins, a energy source and various supplements. Usually serum or serum replacement is used as supplement.

Primary cells are often preferred to established cell lines as they better represent the cells in vivo in terms of activity and functions. On the other hand, there is significant difference between original culture and present culture and even between cultures of the same cells in different laboratories. The rate of growth increases and there is change in chromosome numbers of lines over the time. The differences in phenotypes can cause problems in comparison and interpretation of results. The advantage of cell lines are ease handling, grow continuously, large yield and readily available. Primary cell cultures compared to established cell line are more fastidious in their growth requirements because many cell lines are derived from tumour tissue and demonstrate reduced growth factor requirements.

Cell growth and cell passage : Different types of cells have different growth requirements but in all cases the surrounding medium must supply all the essential nutrients. The temperature, pH, osmolarity and humidity must be kept within limits. Again, toxic or inhibitory substances must not be allowed to accumulate. Serum, a source of macromolecular growth factors, is essential to the growth of many cell types. Anchorage dependent cells require nontoxic or biologically inert glass or plastic surface for attachment.

In may cases, it becomes necessary to send the cells from one laboratory to another laboratory within or outside the country. The ideal method is to fill the flask containing cells at log/exponential phase with growth medium, tightly close the cap, seal it with parafilm and tape and pack it properly with polythene containing tissue paper and cotton. The tissue paper and cotton is used to soak the medium in case of accidental breakage. The polythene containing tissue culture flask having the adherent cell sheet should again be packed with thick paper. Finally, it is to be kept in a wooden/metal box or container.

Vero (monkey, African green, kidney) 
Morphology: Fibroblast
Species:, African green monkey 
Tissue: Kidney

Properties: Virology, virus titration, virus replication, plaque assays, bacterial cytotoxicity

If the concentration of cells in suspension cell culture has reached to 7X10⁵ cells/ml, it is to be subcultured. Suspension cells are diluted to concentration of approximately 1X10⁵ cells/ml although it varies from cell line to cell line. So, to subculture into a 3 litre glass flask using 500 ml of suspension of cells, there is a need of 5X10⁷ cells. As the concentration of cells is 7X10⁵ cells/ml, there is a need of 71.4 ml of cells. Adherent cells are put in the culture flasks at a concentration of 5X10⁴ cells in a 25 cm² flask. For example, after trypsinization, the cells have been resuspended in 5 ml of medium and cell concentration is 1X10⁵ cells/ml or total number is 5X10⁵ cells. If one wants to put cells into two 25 cm² flasks each containing 5X10⁴ cells in 5 ml volume, there is a need of 1X10⁵ cells, which can be obtained from one ml of cell suspension (1X10⁵ cells/ml).

Small volume cultures are useful for experiments such as studying cell morphology, titration of viruses, plaque assay, pock assay, SNT and to compare the effect of agents on growth and metabolism. However, there are other application where there is a need of large number of cells such as extraction of DNA/RNA from cells, production of viruses for vaccine production and a number of products namely interferons, interleukins, hormones, enzymes, antibodies, glycoproteins etc. The large quantity of cells can be obtained by using a large number of flasks/bottles but it is tedius, labour intensive, expensive and there is a risk of contiamination. Although the unit processes are more cost effective and efficient, it needs some modification to overcome limiting factors such as oxygen depletion, shear damage and metabolic toxicity. There are a number of aberrations available to scale up cell culture described below.

There are a number of different cell lines derived from human and animals. With the dramatic increase in numbers of cell lines, the risk of intraspecies and interspecies cross contamination rises proportionally. It is particularly a major problem in laboratories where many different cell lines of human and animal origin are handled at a time. The purity of the primary cell or continuous cell lines and species of its origin should be checked on a regular basis to authenticate the cell line identity. In the absence of such monitoring, inter and intra species cell line contamination are likely to occur in the laboratory resulting in loss of investigator’s time effort and resources. The advantages of working with a well defined cell line free from contaminating organisms would appear obvious.

Early passage cell lines are relatively unstable and go through a period of adaptation to culture. However, between 5^th and 35^th generation (human diploid fibroblasts), the culture is fairly consistent although it varies from one cell to another cell. Finite cell lines should be preserved after adaptation but before senescence (getting older). Cells should be preserved to protect cell line instability, availability in case of contamination, incubator failure or accidental loss.

Cell culture contamination continues to be a major problem and a subject of great concern. Contamination may enter the cell culture system as physical, chemical or biological component of the environment and biological contamination represent the greatest threat as living organism metabolize and replicate. Replication increases the titre of the contaminant.
 
Physical contamination : Physical components of cell culture systems such as radiation, irradiation (UV light and fluorescent light), temperature extremes can cause reduced cell growth and cell death. To minimize the problems, incubators should be located where ambient temperature is relatively constant and no vibration (near centrifuge). Media and media components should not be stored in glass door refrigerators, next to radioisotopes, bench tops or outside the refrigerator or in the hood for prolonged period.

Enumeration of cells is essential to determine the multiplicity of infection, efficiency of plating and to obtain the specific number of cells for an experiment. The concentration of cells of a suspension can be counted using a haemocytometer. Each chamber is divided into a grid of nine large squares. Each large square is 1mmX1mm (area 1 mm²) and the depth is o.1 mm. The volume of each square is 0.1 mm³ or 10^-4 cm³. Since 1 cm³ is equal to 1 ml, the number of cells per ml of suspension can be obtained by multiply the average count per large square by 10⁴ and reciprocal of dilution factor. Dilution is made in such a way that one large square contain about 20-60 cells. The haemocytometer chambers should be filled by capillary action and cell clump are counted as one cell. A total count does not distinguish between living and dead cells. The number of viable cells can be determined by staining the cells with trypan blue or erythrosine B. In trypan blue staining cells appear as unstained whereas dead cells appear as blue.

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

The most important property of a virus is its infectivity or ability to invade a cell and parasitize that cell to replicate itself. To measure infectivity, a titre is defined as the given number of infectious virus units/unit volume and an infectious unit is the smallest amount of virus that produces some recognizable effect in the host system. There are two basic types of infectivity assays (a) Quantal assays and (b) Quantitative assays.

Quantal assays do not measure the number of infectious virus particles present in the inoculum but denote a value for the virus titre where measurement is based on an all or none principle, CPE + or -, dead or alive etc. In the quantitative assays, the number of virus particles present in the suspension can be quantified.

The tentative diagnosis of viral diseases of livestock can be made by clinical symptoms alone. The clinical diagnosis is substantiated by the laboratory to obtain a precise, correct and prompt diagnosis. The success however depends on the collection of suitable materials in proper transport medium at a particular stage of disease condition and its dispatch to the laboratory in a manner that little or no alterations occurs in the specimen. This would facilitate the isolation and identification of the causative viral agents easily.

Bovine
 
1.    PPR    Eye, mouth, rectal swab in PBS on ice, 10 ml blood  in anti-coagulant at height of temperature, Prescapular lymph node, spleen, piece of intestine on ice, lung, liver, spleen, tonsil in 10% neutral formol saline solution.

Processing

On arrival in the laboratory, the specimens are processed immediately or refrigerated until processed. For inoculation into animal/cell culture/embryonated eggs (EE), swabs are shaken in fluid medium, faeces are dispersed in fluid medium and tissue specimens are homogenized in a high speed blender/pestle and mortar with the help of an abrasive like powdered glass or sand. Good quality hard sand should be properly washed in distilled water, treated with HCl, washed thoroughly, dried and sterilized before use. Generally the infective tissue is homogenized in a few ml of buffer usually having a pH of 7.2 to 7.3 to make about 10% tissue suspension. The suspension is centrifuged at about 1000g for 10-15 minutes to sediment the tissue debris and coarse materials.

Principle

The infectivity of a virus may be neutralized by specific antibody when they are mixed and incubated. In some cases, the addition of homologous or guinea pig complement to the mixture enhances neutralization, particularly when early antiserum is used. Serum must be inactivated by heating at 560C for 30 minutes to inactivate complement or to remove non-specific virus inhibitors. Serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the only virus controls develop cytopathic effects. Antibody by neutralizing the infectivity of the virus, protects the cells against virus destruction.

Early and accurate diagnosis of disease is of prime importance for both the successful treatment of the patient and in limiting the spread of disease in the community. It is in this context that immunodiagnostic methods are now being used extensively since they can be extremely sensitive and highly specific. Among the most successful methods are those utilizing labelled antibodies or antigens. Examples of these are immunofluorescence in which antibodies labelled with fluorescent dyes are utilized and radioimmunoassay in which antigens or antibodies are labeled with isotopes. Both of these procedures have proved to be of great value for particular purposes.