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A heart (or any other organ) is comprised of a group of cells. To the best of my knowledge, the growth of a heart depends on cell division. However, cell division by itself doesn't seem to explain why heart cells collectively manifest the form of a heart.
What causes tissues to manifest the various forms that they do?
This is a subject of active research. All the cells in the zygote are identical up to 8 cell stage. At the next division resulting in 16 cells formation it is called a morula.
The zygote has totipotent cells meaning each cell has the ability to develop into full organism by itself if it separates from the zygote (this is how identical twins are born).
Human zygotes are totipotent till at least 4 cell stage. In other primates experiments have demonstrated totipotency upto 16 cell stage.
The totipotent cells differentiate into pluripotent cells and then into various distinct cells belonging to different systems. This process is very complex and a lot of embryological factors and chemicals come into play.
The embryonic factors cause migration (called embryotaxis) and differentiation of the embryonic cells. Some of the factors are sonic hedgehog, wnt, insulin like growth factors, Hox, etc…
Different factors cause differentiation into different cell lines. These factors also cause the organs to attain specific shape and structure, thus the heart is shaped as a heart and the liver is shaped as a liver and so on.
Once the cells differentiate into a particular line, the stem cells of that region generally differentiate into that particular line due to the paracrine influence in that locus, even though these stem cells can differentiate into other cell lines when appropriately stimulated (thus demonstrating pluripotency). So intestinal stem cells would differentiate into goblet cells, surface epithelial cells, Enterochromaffin cells, etc… which are present in that locus.
For more see here:
Dry gangrene symptoms include:
- Shriveled skin that changes from blue to black and eventually comes off
- Cold, numb skin
Symptoms of wet gangrene include:
- Swelling and pain and feeling unwell
- Red, brown, purple, blue, greenish-black, or black skin or sores with a bad-smelling discharge (pus)
- A crackling noise when you press on the affected area
- Thin, shiny, or hairless skin
- A line between healthy and damaged skin
Internal gangrene causes severe pain in the affected area. For example, if you have gangrene in your appendix or colon, you’d probably have belly pain. Internal gangrene can also cause a fever.
The Major Endocrine Glands
Located at the base of the brain, the pituitary gland produces many hormones that regulate other organs. Because of this, the pituitary is often referred to as the "master" endocrine gland, although the term ⋎ntral" endocrine gland is more correct because hormone release by the pituitary is primarily regulated by a brain structure called the hypothalamus, which acts to connect the nervous system to the endocrine system. The hypothalamus produces hormones that stimulate or inhibit the release of pituitary hormones. The hypothalamus also produces antidiuretic hormone, which regulates water balance in the body by inhibiting urine formation by the
Hormones released by the pituitary include growth hormone, which increases during childhood and stimulates the growth of muscle, bone, and other tissues. Sporadic bursts in growth hormone release often result in rapid growth "spurts" associated with adolescence. Hyposecretion of growth hormone can result in dwarfism, whereas hypersecretion of growth hormone can cause gigantism and other disorders. The pituitary also produces follicle-stimulating hormone and luteinizing hormone, which stimulate gamete production and sex steroid production in male and female reproductive organs, and prolactin, which stimulates milk formation in the mammary glands.
Located adjacent to the larynx , the thyroid gland primarily produces thyroxine and triiodothyronine, collectively referred to as thyroid hormone. Thyroid hormone stimulates growth of muscles and bones, carbohydrate metabolism, and basal metabolic rate. Its production requires iodine the lack of dietary iodine causes goiter, a thyroid gland that is overly enlarged in an effort to compensate for the thyroid hormone deficiency.
Effects of thyroid disorders in children and adults can differ widely. For example, hyposecretion of thyroid hormone in infants causes congenital hypothyroidism, a disease characterized by mental retardation and poor body growth hyposecretion in adults produces myxedema, with symptoms such as lethargy , weight gain, and dry skin. Conversely, hypersecretion of thyroid hormone in adults causes Graves' disease, a condition characterized by weight loss, nervousness, and dramatic increases in body metabolism. The thyroid also produces calcitonin, a hormone that regulates blood calcium concentration.
The adrenal glands are small organs on the apex of each kidney. The outer layers of cells in the adrenal gland, called the adrenal cortex, produce several hormones that affect reproductive development mineral balance fat, protein, and carbohydrate balance and adaptation to stress. The inner part, called the adrenal medulla, secretes epinephrine and norepinephrine, which activate the sympathetic nervous system and stimulate the ȯight-or-flight" response that helps the body cope with stressful situations, such as fear.
The pancreas produces insulin and glucagon, which function in opposing fashion to regulate blood sugar (glucose) concentration. When blood glucose level rises𠅏or example, after eating a sugar-rich meal—insulin lowers it by stimulating glucose storage in liver and muscle cells as long chains of glucose called glycogen . Conversely, between meals, blood glucose level decreases. In response, the pancreas releases glucagon, which stimulates glycogen breakdown and subsequent release of glucose into the bloodstream. One of the most well characterized endocrine disorders is diabetes mellitus, resulting from hyposecretion of insulin or, more commonly, target cell insensitivity to it.
Endocrine functions of the gonads are addressed in articles on the male and female reproductive systems. The sex hormone testosterone regulates sperm production in males. Estrogen and progesterone influence egg maturation and release (ovulation) and control the uterine (menstrual) cycle in females.
Although the many hormones produced by human endocrine organs have a wide variety of actions, the common purpose of all hormones is to facilitate organ-to-organ communication necessary for body physiology.
Tissue Structure and Components
The structure of tissues varies according to the type of tissue. Remember that tissues are a group of cells that come together to carry out a special function for the body there may be different types of cells that may come together to carry out a special function. In some tissues, the cells may be similar and in some, the cells may be different.
When cells come together, they do so within a confined space called the Extracellular Matrix or the Extracellular Space. You can visualize the Extracellular matrix or space as a community that has many houses and each house as a cell (each house may be similar but they perform special functions such as using some houses as rooms for sleeping, other houses are used as shops while others are used as offices). The Extracellular space is as the community where the houses are built on the land. In tissues, the extracellular matrix holds the different types of cells and provides a n enabling environment for the cells to perform their functions.
The reason why it is called the extra–cellular space or matrix is because the space is located outside the cells (extra cellular space). Even within the extracellular space, there are various proteins that form this space. This will be discussed while describing this space in details.
Therefore, a tissue can be said to have 2 major components the cells and the extracellular matrix.
What causes aging?
There are few physical differences among a group of first graders. But if you check out the same group 65 years later, their physical differences outnumber their similarities. Some will be the epitome of health, while others will be managing one or more chronic conditions. Some will be vigorous, while others will be lethargic.
As we get older, we become physically less like our peers. That's because we are the sum of our life experiences. At age six, not too much has happened to our bodies to make us radically different from our peers. But by middle and old age, we've had decades to develop and maintain habits that have an impact on our health, both negatively and positively.
The environment, too, affects our health, including where we work and live and how much exposure we have to infectious diseases. Aging is universal, but each of us experiences it in different ways.
Aging may be inevitable, but the rate of aging is not. Why and how our bodies age is still largely a mystery, although we are learning more and more each year. Scientists do maintain, however, that chronological age has little bearing on biological age. The number of candles on your birthday cake merely serves as a marker of time it says little about your health.
But which affects us more - our genes or our lifestyles? Find out on the next page.
Aging Causes: Nature or Nurture?
The complexities of getting older make it difficult to pinpoint why one person ages well while another looks and acts older than his years. Are good health and fortitude passed down like blue eyes and blond hair? Or are they a product of the environment, including the food you eat, whether you have been exposed to harmful chemicals or infectious diseases, and how much you exercise? Both certainly play a role, but we don't yet know which has a more powerful influence.
Genes are powerful predictors of health and longevity as well as disease and death, but they're only part of the story. If your parents and grandparents lived well into their nineties, chances are you will, too -- but not if you abuse your body along the way. (Scientists say all genetic bets are off once you've made it to age 80, however. After that, family history has little bearing on longevity.)
And if your father died young of a heart attack or your mother had breast cancer, you may be genetically predisposed to those diseases. Scientists on the Human Genome Project are continually discovering more genetic determinants of chronic and fatal diseases.
While genes partially determine who will develop chronic conditions that hasten the aging process, such as cancer and heart disease, there is no question that a healthy lifestyle is your weapon against the genes you've been dealt, or your ace in the hole if you've got good genes.
A man whose father and brothers died from heart disease in their forties and fifties may very well escape the same fate by exercising regularly and keeping his blood cholesterol levels and body weight in check. On the other hand, a man with no genetic predisposition to heart disease can certainly create heart problems by eating a high fat, artery-clogging diet and leading an entirely sedentary lifestyle.
Healthy living delays many of the body changes that aging brings. And it's never too late to start on the road to better health. Eating a nutritious diet goes a long way toward insuring good health. For instance, getting enough calcium and vitamin D at any age will retard the onset, and the progression, of osteoporosis, a bone disease that causes pain, fractures, hospitalization, and even death in the elderly.
If you're a smoker and you quit at any time, you decrease the chances of having a heart attack. And exercising or becoming more physically active improves lung function and lowers the risk for heart attack, no matter how old you are.
So what changes do your cells, tissues and body systems go through as you age? On the next page, we'll the biological process of aging.
Aging Biology: How do cells age?
Cells, the most basic body unit, are at the center of any discussion about aging. You have trillions of cells, and they're organized into different tissues that make up organs, such as your brain, heart, and skin.
Some cells, such as those that line the gastrointestinal tract, reproduce continuously others, such as the cells on the inside of arteries, lie dormant but are capable of replicating in response to injury. Still others, including cells of the heart, nerves, and muscles, cannot reproduce. Some of these non-reproducing cells have short life spans and must be continually replaced by other cells in the body. (Red and white blood cells are examples.)
Others, such as heart and nerve cells, live for years or even decades. Over time, cell death outpaces cell production, leaving us with fewer cells. As a result, we are less capable of repairing wear and tear on the body, and our immune system is compromised. We become more susceptible to infections and less proficient at seeking out and destroying mutant cells that could cause cancerous tumors. In fact, many older adults succumb to conditions they could have resisted in their youth.
Though cell death is the basis for understanding the aging process, it is not the only factor. The aging process is incredibly complicated, and it's often difficult to distinguish between changes that are the result of time marching on and those that come with common medical conditions, including high blood pressure and heart disease.
Aging is the inevitable decline in the body's resiliency, which ultimately leads to dwindling powers, both mental and physical. Some aging changes affect us all. For example, diminished eyesight that necessitates reading glasses is considered normal, primarily because it affects everyone who lives long enough.
On the other hand, cataracts, which are formations on the lens of the eye that cloud your vision, can be prevented and are not considered part of the aging process, despite their prevalence in older people. To further complicate matters, organs age at different speeds. That's why a 50-year-old may hear as well as someone twenty years his junior, but may have arthritis or high blood pressure.
Theories abound about the underlying cause of aging. Some maintain that aging is preprogrammed into our cells, while others contend that aging is primarily the result of environmental damage to our cells. Although none of the theories can fully explain the process, they do help us better understand how we age. On the next page, we'll explore the most popular aging theories.
Aging Theories: Genes vs. Lifestyles
What's that sound? According to this theory, it's your biological clock, ticking away at a predetermined rate. This theory says that DNA, the cells' genetic material, holds the key to your planned demise from day one. While this aging theory appears fatalistic on the surface, remember that biology is not destiny. You can't change your genes, but you can slow the march of time with better nutrition and regular physical activity.
Your body produces hormones that help regulate myriad functions, including growth and behavior, reproduction, and immune function. In your youth, hormone production is high, but as you get older, hormone levels drop off, causing declines in the body's ability to repair itself and to keep functioning in top form.
Working cells produce waste. Over time, cells make more waste than they can possibly get rid of, which may wreak havoc on their ability to function and slowly lead to their death. Lipofuscin, or age pigment, is one of the waste products found primarily in some nerve and heart-muscle cells. Lipofuscin binds fat and proteins together in the cells. It accumulates over time and may interfere with cell function.
The protein collagen is at the heart of this theory. Collagen, akin to the body's glue, is one of the most common proteins making up the skin, bones, ligaments, and tendons. When we're young, collagen is pliable. But with age, collagen becomes more rigid, and it shrinks. That's why your skin is less elastic than before.
Aesthetics aside, cross-linking may block the transport of nutrients into cells as well as obstruct waste-product removal. Free radicals are destructive marauders roving your body, ready to pounce on healthy cells. They are produced as part of the millions of chemical reactions your body performs to sustain life.
Your body also makes them in response to environmental toxins such as excessive amounts of unprotected sunlight and cigarette smoke. Free radicals oxidize your cells (think rusting metal). As unbalanced, volatile oxygen molecules, they sacrifice healthy cells to make themselves more stable.
In doing so, free radicals destroy or alter DNA, the cell's genetic blueprint, and disrupt many other cell functions. Free radicals may kill cells as a result of their marauding, or they may give rise to mutant cells that can lead to chronic conditions including cancer and heart disease. Fortunately, the body maintains a sophisticated defense system against free radicals. Unfortunately, our defenses wane with time, and cell damage ensues.
This theory could also be called The Use It and Lose It Theory. The idea is that use, and overuse, of your organs pushes them to the brink of destruction. A poor diet, too much alcohol, and cigarette smoking are thought to accelerate natural wear and tear. With age, the body is less able to repair itself.
How does wear and tear occur? Free radicals, which inflict cellular damage, may be culpable. Similar to the wear and tear idea, this theory says you are born with a certain amount of energy. If you live "fast," you die young, because you use up your energy reserves sooner. "Laid-back people," who suffer from less stress and take life easier, would live longer should this theory prove correct.
A strong immune system is your body's most important defense against germs and toxins. White cells engulf and destroy potential pests such as bacteria and viruses. And they manufacture antibodies, the "soldiers" that patrol the bloodstream, attacking and disarming any substance they don't recognize as the body's own.
Problem is, the immune system becomes less efficient with time, and fewer antibodies are produced, increasing your infection risk. What's more, the body may turn on itself by producing antibodies that destroy its own tissue. When that happens, autoimmune disease, such as lupus and rheumatoid arthritis, is the result.
Although we still do not completely understand the process of aging, we do know quite a lot about it, as we've seen. To find out more about the aging process, check out the links on the next page.
Plant Tissue Culture: Benefit, Structure, Types and Techniques
Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various parts of the plants (organs, embryos, tissues, single cells, protoplasts).
The cultivation process is invariably carried out in a nutrient culture medium under aseptic conditions.
Plant cells have certain advantages over animal cells in culture systems. Unlike animal cells, highly mature and differentiated plant cells retain the ability of totipotency i.e. the ability of change to meristematic state and differentiate into a whole plant.
Benefits of Plant Tissue Culture:
Plant tissue culture is one of the most rapidly growing areas of biotechnology because of its high potential to develop improved crops and ornamental plants. With the advances made in the tissue culture technology, it is now possible to regenerate species of any plant in the laboratory.
To achieve the target of creating a new plant or a plant with desired characteristics, tissue culture is often coupled with recombinant DNA technology. The techniques of plant tissue culture have largely helped in the green revolution by improving the crop yield and quality.
The knowledge obtained from plant tissue cultures has contributed to our understanding of metabolism, growth, differentiation and morphogenesis of plant cells. Further, developments in tissue culture have helped to produce several pathogen-free plants, besides the synthesis of many biologically important compounds, including pharmaceuticals. Because of the wide range of applications, plant tissue culture attracts the attention of molecular biologists, plant breeders and industrialists.
Basic Structure and Growth of a Plant:
An adult plant basically consists of a stem and a root, each with many branches (Fig. 42.1). Both the stem and root are characterized by the presence of apical growth regions which are composed of meristematic cells. These cells are the primary source for all the cell types of a plant.
The plant growth and development occur in two different ways:
This is characterized by ceasation of growth as the plant parts attain certain size and shape, e.g., leaves, flowers, fruits.
2. Indeterminate growth:
This refers to the continuous growth of roots and stems under suitable conditions. It is possible due to the presence of meristems (in stems and roots) which can proliferate continuously. As the seed germinates and seedling emerges, the meristematic cells of the root apex multiply. Above the root apex, the cells grow in length without multiplication.
Some of the elongated cells of the outer layer develop into root hairs to absorb water and nutrients from the soil. As the plant grows, root cells differentiate into phloem and xylem. Phloem is responsible for the absorption of nutrients while xylem absorbs water.
The meristematic cells of the shoot apex divide leading to the growth of stem. Some of the stem cells differentiate and develop into leaf primordia, and then leaves. Axillary buds present between the leaf primordia and elongated stem also possess meristems which can multiply and give rise to branches and flowers.
A diagrammatic view of a plant and a flower are respectively depicted in Fig. 42.1 and Fig. 42.2.
Conventional Plant Breeding and Plant Tissue Culture:
Since the time immemorial, man has been closely involved in the improvement of plants to meet his basic needs. The conventional methods employed for crop improvement are very tedious and longtime processes (sometimes decades). Further, in the conventional breeding methods, it is not possible to introduce desired genes to generate new characters or products.
With the developments in plant tissue culture, it is now possible to reduce the time for the creation of new plants with desired characteristics, transfer of new genes into plant cells and large scale production of commercially important products.
Terms Used in Tissue Culture:
A selected list of the most commonly used terms in tissue culture are briefly explained
An excised piece of differentiated tissue or organ is regarded as an explant. The explant may be taken from any part of the plant body e.g., leaf, stem, root.
The unorganized and undifferentiated mass of plant cells is referred to as callus. Generally, when plant cells are cultured in a suitable medium, they divide to form callus i.e., a mass of parenchymatous cells.
The phenomenon of mature cells reverting to meristematic state to produce callus is dedifferentiation. Dedifferentiation is possible since the non- dividing quiescent cells of the explant, when grown in a suitable culture medium revert to meristematic state.
The ability of the callus cells to differentiate into a plant organ or a whole plant is regarded as re-differentiation.
The ability of an individual cell to develop into a whole plant is referred to as cellular totipotency. The inherent characteristic features of plant cells namely dedifferentiation and re-differentiation are responsible for the phenomenon of totipotency. The other terms used in plant tissue culture are explained at appropriate places.
Brief History of Plant Tissue Culture:
About 250 years ago (1756), Henri-Louis Duhamel du Monceau demonstrated callus formation on the decorticated regions of elm plants. Many botanists regard this work as the forward for the discovery of plant tissue culture. In 1853, Trecul published pictures of callus formation in plants.
German botanist Gottlieb Haberlandt (1902), regarded as the father of plant tissue culture, first developed the concept of in vitro cell culture. He was the first to culture isolated and fully differentiated plant cells in a nutrient medium. During 1934-1940, three scientists namely Gautheret, White and Nobecourt largely contributed to the developments made in plant tissue culture.
Good progress and rapid developments occurred after 1940 in plant tissue culture techniques. Steward and Reinert (1959) first discovered somatic embryo production in vitro. Maheswari and Guha (1964) from India were the first to develop anther culture and poller culture for the production of haploid plants.
Types of Culture:
There are different types of plant tissue culture techniques, mainly based on the explant used (Fig. 42.3).
This involves the culture of differentiated tissue from explant which dedifferentiates in vitro to form callus.
Culture of isolated plant organs is referred to as organ culture. The organ used may be embryo, seed, root, endosperm, anther, ovary, ovule, meristem (shoot tip) or nucellus. The organ culture may be organized or unorganized.
Organized organ culture:
When a well-organized structure of a plant (seed, embryo) is used in culture, it is referred to as organized culture. In this type of culture, the characteristic individual organ structure is maintained and the progeny formed is similar in structure as that of the original organ.
Unorganized organ culture:
This involves the isolation of cells or tissues of a part of the organ, and their culture in vitro. Unorganized culture results in the formation of callus. The callus can be dispersed into aggregates of cells and/or single cells to give a suspension culture.
The culture of isolated individual cells, obtained from an explant tissue or callus is regarded as cell culture. These cultures are carried out in dispension medium and are referred to as cell suspension cultures.
Plant protoplasts (i.e., cells devoid of cell walls) are also used in the laboratory for culture.
Basic Technique of Plant Tissue Culture:
The general procedure adopted for isolation and culture of plant tissues is depicted in Fig. 42.4
The requisite explants (buds, stem, seeds) are trimmed and then subjected to sterilization in a detergent solution. After washing in sterile distilled water, the explants are placed in a suitable culture medium (liquid or semisolid form) and incubated. This results in the establishment of culture. The mother cultures can be subdivided, as frequently as needed, to give daughter cultures.
The most important aspect of in vitro culture technique is to carry out all the operations under aseptic conditions. Bacteria and fungi are the most common contaminants in plant tissue culture. They grow much faster in culture and often kill the plant tissue.
Further, the contaminants also produce certain compounds which are toxic to the plant tissue. Therefore, it is absolutely essential that aseptic conditions are maintained throughout the tissue culture operations. Some of the culture techniques are described here while a few others are discussed at appropriate places.
Applications of Plant Tissue Cultures:
Plant tissue cultures are associated with a wide range of applications—the most important being the production of pharmaceutical, medicinal and other industrially important compounds.
In addition, tissue cultures are useful for several other purposes listed below:
1. To study the respiration and metabolism of plants.
2. For the evaluation of organ functions in plants.
3. To study the various plant diseases and work out methods-for their elimination.
4. Single cell clones are useful for genetic, morphological and pathological studies.
5. Embryonic cell suspensions can be used for large scale clonal propagation.
6. Somatic embryos from cell suspensions can be stored for long term in germplasm banks.
7. In the production of variant clones with new characteristics, a phenomenon referred to as soma clonal variations.
8. Production of haploids (with a single set of chromosomes) for improving crops.
9. Mutant cells can be selected from cultures and used for crop improvement.
10. Immature embryos can be cultured in vitro to produce hybrids, a process referred to as embryo rescue.
Callus is the undifferentiated and unorganized mass of plant cells. It is basically a tumor tissue which usually forms on wounds of differentiated tissues or organs. Callus cells are parenchymatous in nature although not truly homogenous. On careful examination, callus is found to contain some quantity of differentiated tissue, besides the bulk of non-differentiated tissue.
Callus formation in vivo is frequently observed as a result of wounds at the cut edges of stems or roots. Invasion of microorganisms or damage by insect feeding usually occurs through callus. An outline of technique used for callus culture, and initiation of suspension culture is depicted in Fig. 42.5.
Explants for callus culture:
The starting materials (explates) for callus culture may be the differentiated tissue from any part of the plant (root, stem, leaf, anther, flower etc.). The selected explant tissues may be at different stages of cell division, cell proliferation and organization into different distinct specialized structures. If the explant used possesses meristematic cells, then the cell division and multiplication will be rapid.
Factors Affecting Callus Culture:
Many factors are known to influence callus formation in vitro culture. These include the source of the explant and its genotype, composition of the medium (MS medium most commonly used), physical factors (temperature, light etc.) and growth factors. Other important factors affecting callus culture are — age of the plant, location of explant, physiology and growth conditions of the plant.
A temperature in the range of 22-28°C is suitable for adequate callus formation. As regards the effect of light on callus, it is largely dependent on the plant species-light may be essential for some plants while darkness is required by others.
The growth regulators to the medium strongly influence callus formation. Based on the nature of the explant and its genotype, and the endogenous content of the hormone, the requirements of growth regulators may be categorized into 3 groups
3. Both auxin and cytokinin.
Suspension culture from callus:
Suspension cultures can be initiated by transferring friable callus to liquid nutrient medium (Fig. 42.5). As the medium is liquid in nature, the pieces of callus remain submerged. This creates anaerobic condition and ultimately the cells may die. For this reason, suspension cultures have to be agitated by a rotary shaker. Due to agitation, the cells gets dispersed, besides their exposure to aeration.
Applications of Callus Cultures:
Callus cultures are slow-growth plant culture systems in static medium. This enables to conduct several studies related to many aspects of plants (growth, differentiation and metabolism) as listed below.
i. Nutritional requirements of plants.
ii. Cell and organ differentiation.
iii. Development of suspension and protoplast cultures.
v. Genetic transformations.
vi. Production of secondary metabolites and their regulation.
The first attempt to culture single cells (obtained from leaves of flowering plants) was made in as early as 1902 by Haberlandt. Although he was unsuccessful to achieve cell division in vitro, his work gave a stimulus to several researchers. In later years, good success was achieved not only for cell division but also to raise complete plants from single cell cultures.
Applications of Cell Cultures:
Cultured cells have a wide range of applications in biology.
1. Elucidation of the pathways of cellular metabolism.
2. Serve as good targets for mutation and selection of desirable mutants.
3. Production of secondary metabolites of commercial interest.
4. Good potential for crop improvement.
Cell Culture Technique:
The in vitro cell culture technique broadly involves the following aspects:
1. Isolation of single cells.
2. Suspension cultures growth and sub-culturing.
3. Types of suspension cultures.
4. Synchronization of suspension cultures.
5. Measurement of growth of cultures.
6. Measurement of viability of cultured cells.
The salient features of the above steps are briefly described.
1. Isolation of Single Cells:
The cells employed for in vitro culture may be obtained from plant organs, and from cultured tissues.
Plant leaves with homogenous population of cells are the ideal sources for cell culture. Single cells can be isolated from leaves by mechanical or enzymatic methods.
Surface sterilized leaves are cut into small pieces (< 1 cm 2 ), suspended in a medium and subjected to grinding in a glass homogenizer tube. The homogenate is filtered through filters and then centrifuged at a low speed to remove the cellular debris. The supernatant is removed and diluted to achieve the required cell density.
The enzyme macerozyme (under suitable osmotic pressure) can release the individual cells from the leaf tissues. Macerozyme degrades middle lamella and cell walls of parenchymatous tissues.
From cultured tissues:
Single cells can be isolated from callus cultures (grown from cut pieces of surface sterilized plant parts). Repeated sub-culturing of callus on agar medium improves the friability of callus so that fine cell suspensions are obtained.
2. Suspension Cultures — Growth and Subculture:
The isolated cells are grown in suspension cultures. Cell suspensions are maintained by routine sub-culturing in a fresh medium. For this purpose, the cells are picked up in the early stationary phase and transferred. As the cells are incubated in suspension cultures, the cells divide and enlarge.
The incubation period is dependent on:
Among these, cell density is very crucial. The initial cell density used in the subcultures is very critical, and largely depends on the type of suspension culture being maintained. With low initial cell densities, the lag phase and log phases of growth get prolonged.
Whenever a new suspension culture is started, it is necessary to determine the optical cell density in relation the volume of culture medium, so that maximum cell growth can be achieved. With low cell densities, the culture will not grow well, and requires additional supplementation of metabolites to the medium. The normal incubation time for the suspension cultures is in the range of 21-28 days.
3. Types of Suspension Cultures:
There are mainly two types of suspension cultures — batch cultures and continuous cultures.
A batch culture is a cell suspension culture grown in a fixed volume of nutrient culture medium. In batch culture, cell division and cell growth coupled with increase in biomass occur until one of the factors in the culture environment (nutrient, O2 supply) becomes limiting. The cells exhibit the following five phases of growth when the cell number in suspension cultures is plotted against the time of incubation (Fig. 42.6).
1. Lag phase characterized by preparation of cells to divide.
2. Log phase (exponential phase) where the rate of cell multiplication is highest.
3. Linear phase represented by slowness in cell division and increase in cell size expansion.
4. Deceleration phase characterized by decrease in cell division and cell expansion.
5. Stationary phase represented by a constant number of cells and their size.
The batch cultures can be maintained continuously by transferring small amounts of the suspension medium (with inoculum) to fresh medium at regular intervals (2-3 days). Batch cultures are characterized by a constant change in the pattern of cell growth and metabolism. For this reason, these cultures are not ideally suited for the studies related to cellular behaviour.
In continuous cultures, there is a regular addition of fresh nutrient medium and draining out the used medium so that the culture volume is normally constant. These cultures are carried out in specially designed culture vessels (bioreactors).
Continuous cultures are carried out under defined and controlled conditions—cell density, nutrients, O2, pH etc. The cells in these cultures are mostly at an exponential phase (log phase) of growth.
Continuous cultures are of two types—open and closed.
Open continuous cultures:
In these cultures, the inflow of fresh medium is balanced with the outflow of the volume of spent medium along with the cells. The addition of fresh medium and culture harvest are so adjusted that the cultures are maintained indefinitely at a constant growth rate. At a steady state, the rate of cells removed from the cultures equals to the rate of formation of new cells.
Open continuous culture system is regarded as chemostat if the cellular growth rate and density are kept constant by limiting a nutrient in the medium (glucose, nitrogen, phosphorus). In chemostat cultures, except the limiting nutrient, all other nutrients are kept at higher concentrations. As a result, any increase or decrease in the limiting nutrient will correspondingly increase or decrease the growth rate of cells.
In turbidostat open continuous cultures, addition of fresh medium is done whenever there is an increase in turbidity so that the suspension culture system is maintained at a fixed optical density. Thus, in these culture systems, turbidity is preselected on the basis of biomass density in cultures, and they are maintained by intermittent addition of medium and washout of cells.
Closed continuous cultures:
In these cultures, the cells are retained while the inflow of fresh medium is balanced with the outflow of corresponding spent medium. The cells present in the outflowing medium are separated (mechanically) and added back to the culture system. As a result, there is a continuous increase in the biomass in closed continuous cultures. These cultures are useful for studies related to cytodifferentiation, and for the production of certain secondary metabolites e.g., polysaccharides, coumarins.
4. Synchronization of Suspension Cultures:
In the normal circumstances, the cultured plant cells vary greatly in size, shape, cell cycle etc., and are said to be asynchronous. Due to variations in the cells, they are not suitable for genetic, biochemical and physiological studies. For these reasons, synchronization of cells assumes significance.
Synchronization of cultured cells broadly refers to the organized existence of majority of cells in the same cell cycle phase simultaneously.
A synchronous culture may be regarded as a culture in which the cell cycles or specific phase of cycles for majority of cultured cells occurs simultaneously.
Several methods are in use to bring out synchronization of suspension cultures. They may be broadly divided into physical and chemical methods.
The environmental culture growth influencing physical parameters (light, temperature) and the physical properties of the cell (size) can be carefully monitored to achieve reasonably good degree of synchronization. A couple of them are described
When the suspension cultures are subjected to low temperature (around 4°C) shock synchronization occurs. Cold treatment in combination with nutrient starvation gives better results.
The cells in suspension culture can be selected based on the size of the aggregates, and by this approach, cell synchronization can be achieved.
The chemical methods for synchronization of suspension cultures include the use of chemical inhibitors, and deprivation of an essential growth factor (nutrient starvation). By this approach, the cell cycle can be arrested at a particular stage, and then allowed to occur simultaneously so that synchronization is achieved.
Inhibitors of DNA synthesis (5-amino uracil, hydroxyurea, 5-fluorodeoxypurine), when added to the cultures results in the accumulation of cells at G1 phase. And on removal of the inhibitor, synchronization of cell division occurs.
Colchicine is a strong inhibitor to arrest the growth of cells at metaphase. It inhibits spindle formation during the metaphase stage of cell division. Exposure to colchicine must be done for a short period (during the exponential growth phase), as long duration exposure may lead to mitoses.
When an essential nutrient or growth promoting compound is deprived in suspension cultures, this results in stationary growth phase. On supplementation of the missing nutrient compound, cell growth resumption occurs synchronously. Some workers have reported that deprivation and subsequent addition of growth hormone also induces synchronization of cell cultures.
5. Measurement of Growth of Cultures:
It is necessary to assess the growth of cells in cultures. The parameters selected for the measuring growth of suspension cultures include cell counting, packed cell volume and weight increase.
Although cell counting to assess culture growth is reasonably accurate, it is tedious and time consuming. This is because cells in suspension culture mostly exist as colonies in varying sizes. These cells have to be first disrupted (by treating with pectinase or chromic acid), separated, and then counted using a haemocytometer.
Packed cell volume:
Packed cell volume (PCV) is expressed as ml of pellet per ml of culture. To determine PCV, a measured volume of suspension culture is centrifuged (usually at 2000 x g for 5 minutes) and the volume of the pellet or packed cell volume is recorded. After centrifugation the supernatant can be discarded, the pellet washed, dried overnight and weighed. This gives cell dry weight.
Cell fresh weight:
The wet cells are collected on a pre-weighed nylon fabric filter (supported in funnel). They are washed to remove the medium, drained under vacuum and weighed. This gives the fresh weight of cells. However, large samples have to be used for accurate weights.
6. Measurement of Viability of Cultured Cells:
The viability of cells is the most important factor for the growth of cells. Viability of cultured cells can be measured by microscopic examination of cells directly or after staining them.
Phase contrast microscopy:
The viable cells can be detected by the presence of healthy nuclei. Phase contrast microscope is used for this purpose.
Evan’s blue staining:
A dilute solution of Evan’s blue (0.025% w/v) dye stains the dead or damaged cells while the living (viable) cells remain unstained.
Fluorescein diacetate method:
When the cell suspension is incubated with fluorescein diacetate (FDA) at a final concentration of 0.01%, it is cleaved by esterase enzyme of living cells. As a result, the polar portion of fluorescein which emits green fluorescence under ultraviolet (UV) light is released. The viable cells can be detected by their fluorescence, since fluorescein accumulates in the living cells only.
Culture of Isolated Single Cells (Single Cell Clones):
A clone is a mass of cells, all of them derived through mitosis from a single cell. The cells of the clone are expected to be identical with regard to genotype and karyotype. However, changes in these cells may occur after cloning. Single cells separated from plant tissues under suitable conditions can form clones.
Single cells can be cultured by the following methods:
1. Filter paper raft-nurse tissue technique
4. Bergman’s plating technique.
Filter paper raft-nurse tissue technique:
Small pieces of sterile filter papers are placed on established callus cultures several days before the start of single cell culture. Single cell is now placed on the filter paper (Fig. 42.7A). This filter paper, wetted by the exudates from callus tissue (by diffusion) supplies the nutrients to the single cell. The cell divides and forms clones on the filter paper. These colonies can be isolated and cultured.
A microscopic slide or a coverslip can be used to create a micro-chamber. Sometimes, a cavity slide can be directly used. A drop of the medium containing a single cell is placed in the micro-chamber. A drop of mineral oil is placed on either side of the culture drop which is covered with a coverslip (Fig. 42.7B). On incubation, single cell colonies are formed.
For the culture of single cells by micro-drop method, a specially designed dish (cuprak dish) is used. It has a small outer chamber (to be filled with sterile distilled water) and a large inner chamber with a number of micro-wells (Fig. 42.7C). The cell density of the medium is adjusted in such a way that it contains one cell per droplet.
Bergmann’s plating technique:
Bergmann (1960) developed a technique for cloning of single cells. Now a days, Bergmann’s plating technique is the most widely used method for culture of isolated single cells. This method is depicted in Fig. 42.8 and briefly described hereunder.
The cell suspension is filtered through a sieve to obtain single cells in the filtrate. The free cells are suspended in a liquid medium, at a density twice than the required density for cell plating. Now, equal volumes of melted agar (30-35°C) and medium containing cells are mixed.
The agar medium with single cells is poured and spread out in a petridish so that the cells are evenly distributed on a thin layer (of agar after it solidifies). The petridishes (culture dishes) are sealed with a parafilm and incubated at 25°C in dark or diffused light. The single cells divide and develop into clones. The viability of cells in single clones can be measured by the same techniques that have been described for suspension cultures.
Kingdom Alveolata: Dinoflagellates
Dinoflagellates typically possess distinct shapes due to "frames" of cellulose within their cell walls. Their cell surface is generally ridged with perpendicular grooves that house a pair of flagella (shown left). These flagella, the defining characteristic of this group, beat within their grooves and cause dinoflagellates to rotate as they move forward. The word dinoflagellate is derived from the Greek word dinos, which means "rotation" or "whirling," and the Latin flagellum, which means "whip." Many dinoflagellates are photosynthetic accordingly, they comprise a significant proportion of the phytoplankton that floats near the surface of the ocean, making them a critical component of the food web. Phytoplankton are an essential food resource for many other organisms, ranging from heterotrophic protists to baleen whales and many other organisms in between (most of whom serve as food themselves for creatures at higher trophic levels).
Figure 16. A dinoflagellate. (Click to enlarge) Ceratium tripos.
Not all dinoflagellates are photosynthetic many are heterotrophic. Some of these heterotrophs exploit chloroplasts from photosynthetic protists, becoming autotrophic themselves for a time. Some dinoflagellates live in symbiosis with different species, as parasites in some cases and as mutualists in others.
Some dinoflagellates, such as those in the genus Noctiluca, have the ability to bioluminesce (make their own light). This is accomplished with the compound luciferin, which is the same chemical that makes fireflies glow. Noctiluca floats just under the surface of the ocean, and when individuals number in the millions they can produce spectacular glowing tides (pictured below). The red border at the advancing wave front (tide line) as it washes onto the beach is a real visible glow that is triggered by the tumbling dinoflagellates as they hit the sand. If you walk along the tide line of such a beach, your footprints actually glow with each step when your foot disturbs these bioluminescent protists. How bioluminescence evolved is not completely understood. The Burglar Alarm theory posits that the bioluminescent glow attracts predators of dinoflagellate predators and this allows the glowing protist to escape predation.
Figure 17. A dinoflagellate. (Click to enlarge) Noctiluca scintillans is one dinoflagellate responsible for red tides.
Figure 18. A bioluminescent algal bloom. (Click to enlarge)
This image shows a bloom of bioluminescent Noctiluca scintillans.
ELI5: What are "eye-floaters" and how do they manifest/disappear?
Floaters are deposits . within the eye’s vitreous humour, which is normally transparent. At a young age, the vitreous is transparent, but as one ages, imperfections gradually develop. The common type of floater, which is present in most people’s eyes, is due to degenerative changes of the vitreous humour.
Eye floaters are suspended in the vitreous humour, the thick fluid or gel that fills the eye. . Thus, floaters follow the rapid motions of the eye, while drifting slowly within the fluid. When they are first noticed, the natural reaction is to attempt to look directly at them. . Floaters are, in fact, visible only because they do not remain perfectly fixed within the eye. Although the blood vessels of the eye also obstruct light, they are invisible under normal circumstances because they are fixed in location relative to the retina, and the brain "tunes out" stabilized images due to neural adaptation.
Basically: they are tiny pieces of tissue floating in our eyeballs. They are normal, especially as we get older.
Our brain tunes floaters out when they're still, which is why they ɽisappear'. And, they're easier to see against light backgrounds like the sky.
Like /u/shriekingapples said it is caused by debris in the eye. The retina is the layer of the eye that has photo receptors (rods and cone), which captures light and causes a chemical and electrical reaction to the nerve. Light that "excites" the rods causes us to see monochromatic color or how black and white something is, while "excited" cones gives us high-resolution color. As we age the jelly of the eye, the vitreous gel, can shrink and pull away from the retina. This results in debris from the eye to go into the vitreous gel and appear as floaters in our vision. So it is normal that we may see floaters as we age. However, be careful of sudden appearances of floaters. Retinal tears and retinal holes can also cause floaters and/or flashers to appear as well. If not treated the retinal tears and holes can cause a retinal detachment, which may cause even more floaters to appear. If a retinal tear or hole is found early, a small office procedures like laser retinopexy or retinal cryopexy can be performed. Left untreated your vision and the flasher/floaters can become worse. Ultimately leading to surgery as the only option to fixing a retinal detachment.
Source: I work at an ophthalmologists office who specializes in vitrealretinal surgery.
What causes tissues manifest the various forms that they do? - Biology
Connective tissue is a term used to describe the tissue of mesodermal origin that that forms a matrix beneath the epithelial layer and is a connecting or supporting framework for most of the organs of the body. This lab will focus on the so-called connective tissue proper and cartilage the next lab will focus on bone.
Overview of Connective Tissue
In contrast to epithelia, connective tissue is sparsely populated by cells and contains an extensive extracellular matrix consisting of protein fibers, glycoproteins, and proteoglycans. The function of this type of tissue is to provide structural and mechanical support for other tissues, and to mediate the exchange of nutrients and waste between the circulation and other tissues. These tissues have two principal components, an extracellular matrix and a variety of support cells. These two components will be the focus of this lab.
Most frequently, the different types of connective tissues are specified by their content of three distinguishing types of extracellular fibers: collagenous fibers, elastic fibers, and reticular fibers.
The ground substance is an aqueous gel of glycoproteins and proteoglycans that occupies the space between cellular and fibrillar elements of the connective tissue. It is characterized by a gel-like viscous consistency and is polyanionic. The characteristics of the ground substance determine the permeability of the connective tissue layer to solutes and proteins.
Collagenous fibers consist of types I, II, or III collagen and are present in all types of connective tissue. Collagenous connective tissue is divided into two types, based upon the ratio of collagen fibers to ground substance:
- Loose (areolar connective tissue) is the most abundant form of collagenous connective tissue. It occurs in small, elongated bundles separated by regions that contain ground substance.
- Dense connective tissue is enriched in collagen fibers with little ground substance. If the closely packed bundles of fibers are located in one direction, it is called regular if oriented in multiple directions, it is referred to as irregular. An example of regular dense connective tissue is that of tendons an example of irregular dense connective tissue is that of the dermis.
Reticular fibers are composed of type III collagen. Unlike the thick and coarse collagenous fibers, reticular fibers form a thin reticular network. Such networks are widespread among different tissues and form supporting frameworks in the liver, lymphoid organs, capillary endothelia, and muscle fibers.
Elastic fibers contain the protein elastin, which co-polymerizes with the protein fibrillin. These fibers are often organized into lamellar sheets, as in the walls of arteries. Dense, regular, elastic tissue characterizes ligaments. Elastic fibers are stretchable because they are normally disorganized – stretching these fibers makes them take on an organized structure.
Cells of the Connective Tissue Proper
Although the connective tissue has a lower density of cells than the other tissues you will study this year, the cells of these tissues are extremely important.
Fibroblasts are by far the most common native cell type of connective tissue. The fibroblast synthesizes the collagen and ground substance of the extracellular matrix. These cells make a large amount of protein that they secrete to build the connective tissue layer. Some fibroblasts have a contractile function these are called myofibroblasts.
Chondrocytes and osteocytes form the extracellular matrix of cartilage and bone. More details and chondrocytes can be found later in this laboratory osteocytes will be covered in the Laboratory on Bone.
The macrophage is the connective tissue representative of the reticuloendothelial, or mononuclear phagocyte, system. This system consists of a number of tissue-specific, mobile, phagocytic cells that descend from monocytes - these include the Kupffer cells of the liver, the alveolar macrophages of the lung, the microglia of the central nervous system, and the reticular cells of the spleen. You will encounter each of these later in the course for now, make sure you recognize that they all descend from monocytes, and that the macrophage is the connective tissue version. Macrophages are indistinguishable from fibroblasts, but can be recognized when they internalize large amounts of visible tracer substances like dyes or carbon particles. Macrophages phagocytose foreign material in the connective tissue layer and also play an important role as antigen presenting cells, a function that you will learn more about in Immunobiology.
Mast cells are granulated cells typically found in connective tissue. These cells mediate immune responses to foreign particles. In particular, they release large amounts of histamine and enzymes in response to antigen recognition. This degranulation process is protective when foreign organisms invade the body, but is also the cause of many allergic reactions.
White fat cells are specialized for the storage of triglyceride, and occur singly or in small groups scattered throughout the loose connective tissue. They are especially common along smaller blood vessels. When fat cells have accumulated in such abundance that they crowd out or replace cellular and fibrous elements, the accumulation is termed adipose tissue. These cells can grow up to 100 microns and usually contain once centrally located vacuole of lipid - the cytoplasm forms a circular ring around this vacuole, and the nucleus is compressed and displaced to the side. The function of white fat is to serve as an energy source and thermal insulator.
Brown fat cells are highly specialized for temperature regulation. These cells are abundant in newborns and hibernating mammals, but are rare in adults. They have numerous, smaller lipid droplets and a large number of mitochondria, whose cytochromes impart the brown color of the tissue. The electron transport chain of these mitochondria is disrupted by an uncoupling protein, which causes the dissipation of the mitochondrial hydrogen ion gradient without ATP production. This generates heat.
Cartilage is a specialized form of connective tissue produced by differentiated fibroblast-like cells called chondrocytes. It is characterized by a prominent extracellular matrix consisting of various proportions of connective tissue fibers embedded in a gel-like matrix. Chondrocytes are located within lacunae in the matrix that they have built around themselves. Individual lacunae may contain multiple cells deriving from a common progenitor. Lacunae are separated from one another as a result of the secretory activity of the chondrocytes.
A highly fibrous, organized, dense connective tissue capsule known as the perichondrium surrounds cartilage. The fibroblast-like cells of this layer have chondrogenic potentiality, and are responsible for the enlargement of cartilage plates by appositional growth. Appositional growth involves cell division, differentiation, and secretion of new extracellular matrix, thereby contributing mass and new cells at the cartilage surface. It is in contrast to interstitial growth, in which new matrix is deposited within mature cartilage.
Three kinds of cartilage are classified according to the abundance of certain fibers and the characteristics of their matrix: