Science Links

Green Tea, White Tea: Health Catechin

2006-03-24T01:27:00.000-08:00

Darjeeling Tea Processing - The process of Darjeeling Tea manufacture

2006-03-24T01:25:00.000-08:00

Darjeeling Tea Processing - The process of Darjeeling Tea manufacture

Darjeeling Tea Processing
(Tea Manufacture)

PLUCKING:
During quality periods i.e. first flush or second flush, two leaves and a bud are picked - this is called fine plucking, resulting in high quality teas. At other times, even three or four leaves and a bud are plucked - this is called coarse plucking. The plucking cycle is maintained at about 7 day intervals. The plucked leaves are collected in bamboo baskets, taking care that they are not crushed by overloading the baskets. WEIGHMENT : The plucked leaves are delivered to the factory for weighment. Each plucker is paid against the quantity (weight) of green leaf that he/she brings in.

WEIGHMENT :
The plucked leaves are delivered to the factory for weighment. Each plucker is paid against the quantity (weight) of green leaf that he/she brings in.

WITHERING:
The green leaf, after eradication of any foreign matter, is spread on "withering troughs", loosely, to a depth of 6 inches. Fans are installed to pass air over the green leaf while it withers. The object of the withering process is to get rid of the moisture content in the green leaf and prepare the leaf to withstand the strain of rolling without breaking up. Period of withering can vary from 18 to 24 hrs. depending on the moisture content. The leaf, when properly withered, gives off a fragrant odour.

ROLLING:
The object of rolling is to bruise the cells of the leaves so that their sap (juice) is exposed to the action of oxygen in the air. Rolling also gives a twist to the leaf. The cell sap contains tannins, caffeine, proteins and other chemical substances, which ultimately give the characteristic colour to the tea liquor during infusion. The withered leaf is given 3 or 4 rolls (each of 30 minutes). After the first roll, the leaf is sifted (kutcha sifting) and the fine leaves (about 20%) are taken out. The rest are given a second roll and in the second kutcha sifting about 20 - 25% rolled leaf is taken out. This process is repeated on the remainder with a 3rd or 4th roll. The first roll is done at low pressure, second at medium and 3rd at harder pressure.

FERMENTATION:
The rolled leaves are spread on fermenting beds and left to ferment for a period of 3 to 4 hrs. The leaves are loosely spread to a thickness of 1 or 1.5. inches. Good fermentation results in the colour of the leaf to change to reddish brown giving off the characteristic aroma after the juices in the rolled leaves react with each other and the air.

FIRING:
The fermented leaves are then fired (i.e. heated) in a drier machine. The object of this process is to arrest fermentation and slowly dessicate the leaf in such a way so as to extract the moisture without scorching the tea and at the same time, preserving its quality and other characters to the optimum level. The leaves are passed through the driers and remain within the driers for a period of approx. 20 mins, at a temperature of around 240 to 250° F. This results in the leaves moisture content to come down to 20 - 25% from 60 - 70% before it enters the drier. A second firing is also given shortly thereafter.

SORTING:
Sorting of different grades is done by sorting machines which are fitted with wire mesh trays that revolve or vibrate. The tea is passed over wire mesh of varying sizes so that the whole leaf, broken leaf, fannings and dust grades fall at different places. These sorted teas comprise the different grades.

PACKING:
The different grades of tea are then packed into plywood chests / paper sacks lined with aluminium foil inside. Each lot is generally packed in a minimum of 5 chests / sacks or more. The chests / sacks are sealed and the grade name, garden name, lot number (called an invoice), chest number, gross and nett weight, year of manufacture etc. are printed on the chests / sacks with stencils. Lastly, Darjeeling CTM user licence number and Darjeeling "CTM-applied for" are also stencilled onto the tea chests / sacks.

Tea: A Story of Serendipity

2006-03-24T01:15:00.000-08:00

Tea: A Story of Serendipity

As legend has it, one day in 2737 B.C. the Chinese Emperor Shen Nung was boiling drinking water over an open fire, believing that those who drank boiled water were healthier. Some leaves from a nearby Camellia sinensis plant floated into the pot. The emperor drank the mixture and declared it gave one "vigor of body, contentment of mind, and determination of purpose."

Perhaps as testament to the emperor's assessment, tea--the potion he unwittingly brewed that day--today is second only to water in worldwide consumption. The U.S. population is drinking its fair share of the brew; in 1994, Americans drank 2.25 billion gallons of tea in one form or another--hot, iced, spiced, flavored, with or without sugar, honey, milk, cream, or lemon.

A serving of tea generally contains about 40 milligrams of caffeine (less than half as much caffeine as in coffee), but the actual levels vary depending on the specific blend and the strength of the brew. Decaffeinated tea is also available.

Many tea drinkers find the beverage soothing, and folk medicine has long valued it as a remedy for sore throats and unsettled stomachs. Recent studies have shown that certain chemicals in tea called polyphenols may help reduce the risk of far more serious illnesses, including atherosclerosis and some cancers, although the data are not conclusive. (See "Tonic in a Teapot?")

Black, Green and Oolong

Two leaves and a bud at a time--This is the secret of fine tea picking. The work is done chiefly by women, who carry light bamboo baskets strapped to their backs.

Tea comes in black, green and oolong varieties, all produced from the leaves of Camellia sinensis, a white-flowered evergreen. The method of processing the leaf distinguishes the three types. (Herbal teas are made from leaves of other plants. FDA requires that herbal tea labels carry the name of the plant the product derives from, such as chamomile. For more on herbal teas, see "Herbal Teas and Toxicity" in the May 1991 FDA Consumer.)

The traditional method of producing black tea begins with withering. The plucked leaves are placed on shelves called withering racks, where excess moisture is removed. They are then rolled in special machines that release the leaves' enzymes and juices, which give tea its aroma and taste. Next, the leaves ferment in a room with controlled temperature and humidity; finally they are dried in ovens. More recently some processors have forsaken the traditional method to speed production by using machines that finely chop the leaves, thereby cutting the time for withering and fermenting.

Green tea is made by steaming or otherwise heating the leaves immediately after plucking to prevent the fermentation that makes black tea. Then the leaves are rolled and dried.

Oolong tea is fermented only partially--to a point between black and green. While the leaves wilt naturally, enzymes begin to ferment them. Processors interrupt the fermentation by stirring the leaves in heated pans, then rolling and drying them.

Different varieties of Camellia sinensis grow in different geographic areas and produce leaves that vary from a very small China leaf, perhaps one-half to three-quarters of an inch long, to the Assam leaf, which may be 3 or 4 inches long. Certain varieties are better suited than others for a particular processing method. For example, the China leaf from China and Formosa produces the best oolongs.

Scented and spiced teas are made from black tea. "Scented teas look just like any other tea," says FDA chemist and tea expert Robert Dick, "because the scent is more or less sprayed on. They're flavored with just about anything--peach, vanilla, cherry. The spiced teas, on the other hand, usually contain pieces of spices--cinnamon or nutmeg or orange or lemon peel--so you can see there's something in there."

What about orange pekoe? Orange pekoe refers to the size of the tea leaf. Processed tea leaves are sorted into sizes by passing them over screens with different size holes. The largest leaves are orange pekoe, pekoe, and pekoe souchong. The smaller or broken leaves are classified as broken orange pekoe, broken pekoe souchong, broken orange pekoe fannings, and fines (also called "dust").

In brewing, flavor and color come out of the larger leaves more slowly than out of the broken and fine grades. The broken grades, which make up about 80 percent of the total black tea crop, produce a stronger, darker tea. The grades have nothing to do with the quality or flavor of tea; they simply refer to leaf size.

"Technically, except for fannings and fines, the terms should apply only to black, or fermented, tea," Dick says, "but nowadays I often see oolongs labeled "orange pekoe," and even some green teas are labeled pekoe or flowery pekoe."

Tea tastes vary, and one aficionado who squirts lemon in his cup may cringe at the sight of another pouring milk or honey. But no matter how the tea may be doctored, in the United States the odds are overwhelming that it starts out black. Nearly 95 percent of all tea consumed here is black, according to the New York City-based Tea Council of the U.S.A.; 4 percent is green, 1 percent oolong, and 1 percent flavored.

That wasn't always the case, and our proclivity for drinking black tea over green or oolong may have been influenced by events in history. Sixty years ago and more, the amount of black and green tea Americans drank was split fairly evenly--each accounting for about 40 percent of the market--with oolong constituting the rest. During World War II, however, the major sources of green tea--China and Japan--were cut off from the United States, leaving us with tea almost exclusively from British-controlled India, which produces black tea. Americans came out of the war drinking nearly 99 percent black tea.

With the Korean War in the 1950s, uncertainties about tea supplies resurfaced, and the United States began to look for other suppliers.

"Argentina filled the bill," Dick says, "because tea could grow very fast there. Although the country didn't produce an outstanding tea, it produced a good average tea."

Today, most of our tea comes from Argentina, China (which got back into the U.S. market in 1978), and Java. Thirty years ago most of it came from India and Ceylon (now Sri Lanka). Argentine black tea is the kind most used for iced tea, and that's another reason black tea dominates the U.S. market.

pH Water Quality Information

2006-02-12T04:28:00.000-08:00

pH Water Quality Information

The World Carrot Museum

2005-12-29T00:46:00.000-08:00

50 Harmful Effects of Genetically Modified Foods

2005-12-28T01:46:00.000-08:00

MB Technical Manual

2005-12-18T18:22:00.000-08:00

A Science Odyssey: People and Discoveries: Worldwide flu pandemic strikes

2005-12-08T00:31:00.000-08:00

A Science Odyssey: People and Discoveries: Worldwide flu pandemic strikes

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A Science Odyssey
People and Discoveries

Worldwide flu pandemic strikes
1918 - 1919

Late in the spring of 1918 the Spanish wire service Agencia Fabra sent cables of an unusual nature to Reuter's news service headquarters in London. "A strange form of disease of epidemic character has appeared in Madrid," it said. "The epidemic is of a mild nature, no deaths having been reported." The illness began with a cough, then headache and backache, fatigue, high fever, racing heart, loss of appetite and labored breathing. It usually lasted about three days. Cases had cropped up over the spring and summer in other countries, too, from Norway to India, China to Costa Rica. But in Spain, suddenly 8 million people were down with the bug. And as the summer of 1918 turned to fall, the epidemic lost its mildness: people started to die.

The influenza commonly called "Spanish flu" killed more people than the guns of World War I. Estimates put the worldwide death toll at 21,642,274. Some one billion people were affected by the disease -- half of the total human population. It came at a time when 19 nations were at war and the disruption, stress, and privation of war certainly aided the flu's transmission. It killed people on every continent except Antarctica, with the most lives lost in Asia and the highest percentage of population killed in India. From August 1918, when cases of the flu started looking abnormally high, until the following July when they returned to about normal, 20 million Americans became sick and more than 500,000 died. In October, 1918, the flu reached its peak, killing about 195,000 Americans. About 57,000 American soldiers died from influenza while the U.S. was at war; about 53,500 died in battle.

There wasn't much doctors could do. In the course of the epidemic nearly every known therapy was tried -- quinine tablets, bleeding, castor oil, digitalis, morphine, enemas, aspirin, tobacco, hot baths, cold baths, iron tonics, and expectorants of pine tar. Little was known about the virus, except that it was contagious. After deaths from the disease began in earnest, many local governing bodies closed down theaters, churches, and other public gatherings. Ordinances made it illegal to spit, cough, or sneeze in public -- with threat of $500 fines in New York City. When people went out they wore gauze masks over their nose and mouth, often soaked in camphor or other medicinal substances.

After months of terrorizing people around the world, the "Spanish lady" (called "The Naples Soldier" in Spain, and a variety of other names around the world) seemed to withdraw. It had been the most dire epidemic since the Middle Ages, the third worst in recorded history. For all its destruction, it did not get much press at the time. War and then peace monopolized the front pages. And still little is known about the origin or nature of the killer virus. Many believe the modern "swine flu" virus is a descendant of the deadly 1918 flu. Some theorize that its stronger ancestor ganged up with a bacteria to wreak havoc on the human population. In recent years, vaccinations against various strains of influenza have been introduced.

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The 1918 Influenza Pandemic

2005-12-08T00:27:00.000-08:00

The 1918 Influenza Pandemic: "North America, Europe, Asia, Africa, Brazil and the South Pacific"

The Influenza Pandemic of 1918

The influenza pandemic of 1918-1919 killed more people than the Great War, known today as World War I (WWI), at somewhere between 20 and 40 million people. It has been cited as the most devastating epidemic in recorded world history. More people died of influenza in a single year than in four-years of the Black Death Bubonic Plague from 1347 to 1351. Known as "Spanish Flu" or "La Grippe" the influenza of 1918-1919 was a global disaster.

The Grim Reaper by Louis Raemaekers

In the fall of 1918 the Great War in Europe was winding down and peace was on the horizon. The Americans had joined in the fight, bringing the Allies closer to victory against the Germans. Deep within the trenches these men lived through some of the most brutal conditions of life, which it seemed could not be any worse. Then, in pockets across the globe, something erupted that seemed as benign as the common cold. The influenza of that season, however, was far more than a cold. In the two years that this scourge ravaged the earth, a fifth of the world's population was infected. The flu was most deadly for people ages 20 to 40. This pattern of morbidity was unusual for influenza which is usually a killer of the elderly and young children. It infected 28% of all Americans (Tice). An estimated 675,000 Americans died of influenza during the pandemic, ten times as many as in the world war. Of the U.S. soldiers who died in Europe, half of them fell to the influenza virus and not to the enemy (Deseret News). An estimated 43,000 servicemen mobilized for WWI died of influenza (Crosby). 1918 would go down as unforgettable year of suffering and death and yet of peace. As noted in the Journal of the American Medical Association final edition of 1918:

"The 1918 has gone: a year momentous as the termination of the most cruel war in the annals of the human race; a year which marked, the end at least for a time, of man's destruction of man; unfortunately a year in which developed a most fatal infectious disease causing the death of hundreds of thousands of human beings. Medical science for four and one-half years devoted itself to putting men on the firing line and keeping them there. Now it must turn with its whole might to combating the greatest enemy of all--infectious disease," (12/28/1918).

An Emergency Hospital for Influenza Patients

The effect of the influenza epidemic was so severe that the average life span in the US was depressed by 10 years. The influenza virus had a profound virulence, with a mortality rate at 2.5% compared to the previous influenza epidemics, which were less than 0.1%. The death rate for 15 to 34-year-olds of influenza and pneumonia were 20 times higher in 1918 than in previous years (Taubenberger). People were struck with illness on the street and died rapid deaths. One anectode shared of 1918 was of four women playing bridge together late into the night. Overnight, three of the women died from influenza (Hoagg). Others told stories of people on their way to work suddenly developing the flu and dying within hours (Henig). One physician writes that patients with seemingly ordinary influenza would rapidly "develop the most viscous type of pneumonia that has ever been seen" and later when cyanosis appeared in the patients, "it is simply a struggle for air until they suffocate," (Grist, 1979). Another physician recalls that the influenza patients "died struggling to clear their airways of a blood-tinged froth that sometimes gushed from their nose and mouth," (Starr, 1976). The physicians of the time were helpless against this powerful agent of influenza. In 1918 children would skip rope to the rhyme (Crawford):

I had a little bird,
Its name was Enza.
I opened the window,
And in-flu-enza.

The influenza pandemic circled the globe. Most of humanity felt the effects of this strain of the influenza virus. It spread following the path of its human carriers, along trade routes and shipping lines. Outbreaks swept through North America, Europe, Asia, Africa, Brazil and the South Pacific (Taubenberger). In India the mortality rate was extremely high at around 50 deaths from influenza per 1,000 people (Brown). The Great War, with its mass movements of men in armies and aboard ships, probably aided in its rapid diffusion and attack. The origins of the deadly flu disease were unknown but widely speculated upon. Some of the allies thought of the epidemic as a biological warfare tool of the Germans. Many thought it was a result of the trench warfare, the use of mustard gases and the generated "smoke and fumes" of the war. A national campaign began using the ready rhetoric of war to fight the new enemy of microscopic proportions. A study attempted to reason why the disease had been so devastating in certain localized regions, looking at the climate, the weather and the racial composition of cities. They found humidity to be linked with more severe epidemics as it "fosters the dissemination of the bacteria," (Committee on Atmosphere and Man, 1923). Meanwhile the new sciences of the infectious agents and immunology were racing to come up with a vaccine or therapy to stop the epidemics.

The experiences of people in military camps encountering the influenza pandemic:

An excerpt for the memoirs of a survivor at Camp Funston of the pandemic Survivor

A letter to a fellow physician describing conditions during the influenza epidemic at Camp Devens

A collection of letters of a soldier stationed in Camp Funston Soldier

The origins of this influenza variant is not precisely known. It is thought to have originated in China in a rare genetic shift of the influenza virus. The recombination of its surface proteins created a virus novel to almost everyone and a loss of herd immunity. Recently the virus has been reconstructed from the tissue of a dead soldier and is now being genetically characterized. The name of Spanish Flu came from the early affliction and large mortalities in Spain (BMJ,10/19/1918) where it allegedly killed 8 million in May (BMJ, 7/13/1918). However, a first wave of influenza appeared early in the spring of 1918 in Kansas and in military camps throughout the US. Few noticed the epidemic in the midst of the war. Wilson had just given his 14 point address. There was virtually no response or acknowledgment to the epidemics in March and April in the military camps. It was unfortunate that no steps were taken to prepare for the usual recrudescence of the virulent influenza strain in the winter. The lack of action was later criticized when the epidemic could not be ignored in the winter of 1918 (BMJ, 1918). These first epidemics at training camps were a sign of what was coming in greater magnitude in the fall and winter of 1918 to the entire world.

The war brought the virus back into the US for the second wave of the epidemic. It first arrived in Boston in September of 1918 through the port busy with war shipments of machinery and supplies. The war also enabled the virus to spread and diffuse. Men across the nation were mobilizing to join the military and the cause. As they came together, they brought the virus with them and to those they contacted. The virus killed almost 200,00 in October of 1918 alone. In November 11 of 1918 the end of the war enabled a resurgence. As people celebrated Armistice Day with parades and large partiess, a complete disaster from the public health standpoint, a rebirth of the epidemic occurred in some cities. The flu that winter was beyond imagination as millions were infected and thousands died. Just as the war had effected the course of influenza, influenza affected the war. Entire fleets were ill with the disease and men on the front were too sick to fight. The flu was devastating to both sides, killing more men than their own weapons could.

photo

With the military patients coming home from the war with battle wounds and mustard gas burns, hospital facilities and staff were taxed to the limit. This created a shortage of physicians, especially in the civilian sector as many had been lost for service with the military. Since the medical practitioners were away with the troops, only the medical students were left to care for the sick. Third and forth year classes were closed and the students assigned jobs as interns or nurses (Starr,1976). One article noted that "depletion has been carried to such an extent that the practitioners are brought very near the breaking point," (BMJ, 11/2/1918). The shortage was further confounded by the added loss of physicians to the epidemic. In the U.S., the Red Cross had to recruit more volunteers to contribute to the new cause at home of fighting the influenza epidemic. To respond with the fullest utilization of nurses, volunteers and medical supplies, the Red Cross created a National Committee on Influenza. It was involved in both military and civilian sectors to mobilize all forces to fight Spanish influenza (Crosby, 1989). In some areas of the US, the nursing shortage was so acute that the Red Cross had to ask local businesses to allow workers to have the day off if they volunteer in the hospitals at night (Deseret News). Emergency hospitals were created to take in the patients from the US and those arriving sick from overseas.

The pandemic affected everyone. With one-quarter of the US and one-fifth of the world infected with the influenza, it was impossible to escape from the illness. Even President Woodrow Wilson suffered from the flu in early 1919 while negotiating the crucial treaty of Versailles to end the World War (Tice). Those who were lucky enough to avoid infection had to deal with the public health ordinances to restrain the spread of the disease. The public health departments distributed gauze masks to be worn in public. Stores could not hold sales, funerals were limited to 15 minutes. Some towns required a signed certificate to enter and railroads would not accept passengers without them. Those who ignored the flu ordinances had to pay steep fines enforced by extra officers (Deseret News). Bodies pilled up as the massive deaths of the epidemic ensued. Besides the lack of health care workers and medical supplies, there was a shortage of coffins, morticians and gravediggers (Knox). The conditions in 1918 were not so far removed from the Black Death in the era of the bubonic plague of the Middle Ages.

In 1918-19 this deadly influenza pandemic erupted during the final stages of World War I. Nations were already attempting to deal with the effects and costs of the war. Propaganda campaigns and war restrictions and rations had been implemented by governments. Nationalism pervaded as people accepted government authority. This allowed the public health departments to easily step in and implement their restrictive measures. The war also gave science greater importance as governments relied on scientists, now armed with the new germ theory and the development of antiseptic surgery, to design vaccines and reduce mortalities of disease and battle wounds. Their new technologies could preserve the men on the front and ultimately save the world. These conditions created by World War I, together with the current social attitudes and ideas, led to the relatively calm response of the public and application of scientific ideas. People allowed for strict measures and loss of freedom during the war as they submitted to the needs of the nation ahead of their personal needs. They had accepted the limitations placed with rationing and drafting. The responses of the public health officials reflected the new allegiance to science and the wartime society. The medical and scientific communities had developed new theories and applied them to prevention, diagnostics and treatment of the influenza patients.

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Graphs of the Influenza Epidemic Impact

The Public Health Response
Authoritative Measures
Preventative Measures
Prophylaxis

The Scientific and Medical Response
Clinical Descriptions
Treatment and Therapy
The Etiology of Influenza

Bibliography

by Molly Billings, June, 1997 modified RDS February, 2005

Teaching Units: Epidemiology

2005-12-07T23:52:00.000-08:00

NSRL : National Soybean Research Laboratory

2005-12-06T00:04:00.000-08:00

Organic Chemistry Demonstration Experiments on Video - Chemistry Visualized

2005-12-01T20:04:00.000-08:00

Organic Chemistry Demonstration Experiments on Video - Chemistry Visualized

Worksheets, Teaching Tips, Teacher Resources, and Rubrics from TeAch-nology.com

2005-11-23T23:31:00.000-08:00

Worksheets, Teaching Tips, Teacher Resources, and Rubrics from TeAch-nology.com

ecology and science

2005-11-22T21:02:00.000-08:00

Biology 102 Homepage

products to tackle mosq

2005-10-11T20:24:00.000-07:00

mosquito bites, Mosquito Bites, MOSQUITO BITES, mosquito spray, Mosquito Spray, MOSQUITO SPRAY, MOSQUITO CONTROL, mosquito control, mosquito spray, mosquitoes, mosquito, mosquito control, mosqito control, Mosqito Control, MOSQITO CONTROL, mosquitoes, Mosquitoes, mosquit control, Mosquit Control, MOSQUIT CONTROL, mosquito spray, mosquito treating, mosquito prevention

MOSQUITO CONTROL

This article is about MOSQUITO control. It will explain why they are a pest and what needs to be done for controlling infestations. PLEASE NOTE: YOU CAN SEE PICTURES AND PRICING OF ALL THE PRODUCTS LISTED IN THIS ARTICLE BY CLICKING YOUR MOUSE CURSOR WHERE PRODUCTS APPEAR UNDERLINED IN THE TEXT BELOW. Most of your questions will be answered in the article. Be sure to read all of it before you call in for technical support. If you are looking for information about any other insect or animal, go to our article archive section by following the link below where you will find in depth articles and information on just about any pest. CLICK HERE TO GO TO OUR ARTICLE SELECTION PAGE Mosquitoes are a pest that are capable of ruining a great day at the park, a romantic evening on a deck or even spending time in the garden. People will do anything to avoid being bit. They will wear long pants during the summer, use hats with screen veils and even spray themselves with everything imaginable hoping that no mosquito will find them. However, mosquito population's are more active today then ever. If you enjoy the great outdoors, get used to dealing with mosquitoes. They are here to stay and this article will explain methods of control you may use to help diminish their numbers around the home. The author will not get in to great detail about all the species throughout the world. Furthermore, this article is not about diseases - specifically the West Nile Virus - nor detailed mosquito biology. There is some basic information the author will discuss but the focus of this article will be to offer control methods for all kinds of mosquitoes - regardless of species!!! There are over 150 species of mosquitoes in the United States. Some are able to fully develop from eggs in less than a week. Most take 10-14 days to reach maturity but what is important is they grow rapidly. Mosquitoes need water and high levels of moisture to sustain themselves. Although female mosquitoes may live for up to a year, most die in the season they were born. Mosquito populations are able to continue from year to year because one stage is able to overwinter and start their cycle again the next spring. It may be the adult, the pupa, the larva or the egg which is needed. Each species has different winter survivors. Some adult females don't need a blood meal to begin to reproduce. In general, male mosquitoes live a short time. Most mosquitoes lay several hundred eggs and are able to generate huge populations within a short period of time. Although standing water is the prime location for them to reproduce, there are many locations around the home that afford fertile egg laying areas. Such places include water in the bottom of planters, drainage streams, street sewers which don't drain completely, rain barrels, buckets of water, swimming pools, drain lines from rain gutters, old tires, mulch around the home, shrubs, trees, firewood, slow moving water, small decorative ponds for pet fish, bird baths, water accumulating around windows or doors, water accumulating from an automatic sprinkler system, pet water dishes, leaks around water spickets and just about anywhere water is used or is able to accumulate during the warm summer months any where in the country. Mosquitoes need water to reproduce. They will readily move to moist, shady areas under decks, around pools, in garages, in dense shrubbery or flowers, any kind of ivy, holes or nooks of trees, water in a clogged rain gutter or simply the water on a leaf of shrubs which are being watered during the hot summer months. Most people believe mosquitoes are coming from great distances to their yard in search of food. In fact, most mosquitoes migrate to a yard first and foremost because there is something about the yard which the mosquito finds attractive for living. In most cases, mosquitoes are finding a great place to live around the home and then take advantage of the free meals the homeowner or their children present when outside in the yard. Mosquitoes don't migrate far from where they will find shelter and protection from the hot sun. Shade and moisture are two ingredients needed for their survival and can be found around any home. If your home is on a lake or pond, the mosquitoes could be breeding in the water. Generally, they will do so close to shore. Don't expect to find them more than 10 feet from shore. They like shallow water and will keep themselves close to plant life and wet lands if possible. Open deep water which is moving is not the kind of water they like for reproduction. Barns or sheds are another great location for reproduction or shelter. The underside of most decks which are built close to the ground offers great shady shelter and protection for weak mosquitoes susceptible to the hot sun. It is important to locate any area around the home where mosquitoes may be seeking shelter or using for reproduction. The author has experienced a trend in recent years which will only continue. Many homeowners are creating perfect breeding and shelter conditions which are attracting mosquitoes. If you have any of the conditions described above, chances are you will have mosquitoes. Don't be placing the blame on someone else. Mosquitoes will stay where the breeding and shelter areas are best for them. If you are creating a moist shady area around your home, you will be luring mosquitoes. Once they find the shade and moisture to live, expect them to find you and your family for their food! Mosquito control is easy if you are able to determine where they are living or breeding. Inspect around your home and locate where the mosquitoes are most prevalent. Although you may believe they are coming from an adjacent lot, be sure to inspect your property thoroughly. If you have any of the sights listed above, chances are mosquitoes are taking advantage of such conditions. Another way to determine where the mosquitoes are living is to simply stand in certain areas and wait to see how long it takes for them to find you. Mosquitoes will not travel far away from where they are comfortable. The faster you have mosquitoes find you and the more that find you indicate a prime nest or shelter location which needs to be treated. Such "pockets" of mosquitoes exist around most any home and the secret of getting control of the situation is to find as many of these locations. Once found, there are several methods of treatment that can be used. Now that you have determined where to treat, you will need to choose the product best suited for your situation. Mosquito products are designed for treating different types of areas. There are five different product types. First, there are repellents. These are used for repelling mosquitoes from any treated area or from people themselves. Second, there are larvacides. These are products which kill the larva of mosquitoes as they develop. They are usually applied to water where mosquitoes are breeding. Third, there are residual products. These are insecticides which can be applied with a standard pump sprayer or a garden hose end sprayer. Applied to the surfaces where mosquitoes like to land, the residual of these materials will kill mosquitoes as they enter and land on treated surfaces. The fourth type of product used for mosquito control is to directly kill them as they fly. This can be done by aggressively fogging for them or by passively trapping and killing them. Fogging machines offer the most immediate and complete control of all options whereas the newer Mosquito Killing Systems are passive yet somewhat effective. The fifth type of mosquito control that can be employed is a relatively new approach yet one which is both easy and effective. Much like many of the new allergy medicines, there are now Mosquito Blockers. These are devices which affect mosquitoes in such a way they are not able to detect Co2 or octenol. Not being able to detect these compounds prevents them from being able to track a target. Lets examine the products in each category and decide so that you can decide which one will work best for your problem. Repelling mosquitoes have long been the most common method of trying to control local infestations. In fact, there was no real "control" going on; repellents simply push the pest to another area. Citronella, smoke and other compounds have been used over the years but all met with little if any success. However, we now have several repellents that work OK for certain types of applications. The key here is the type of repelling you are looking to achieve. Don't think you will be able to keep mosquitoes away from your yard if they are reproducing or nesting on or adjacent to your land. You don't stand much of a chance for long term repelling under these conditions (as described above). However, you can achieve some temporary relief with these products. They will help to reduce activity around your patio or picnic area so that you can better enjoy the outdoors for a picnic or summer party. MOSQUITO REPELLENT POWDER is a powdery material which can be applied over the turf and plant area where you want to keep mosquitoes away. It is easy to use, will last a week or more (under dry conditions) and poses no hazard to people or animals. In fact, it uses a high PH carrier which conditions the soil much like lime. This product offers short term relief and should not be used with the intention of "controlling" mosquito infestations. Mosquito Repellent is designed to be used right before a party or outdoor get together where a reduction in mosquito activity is desirable. Use some INSECT INCENSE directly around the area as well to aid in the results you will be able to achieve. They are pleasant smelling and non-offensive to people yet mosquitoes and other biting insects don't seem to like being around where they are burning.If you want something a little stronger, set out some of the MOSQUITO COILS. These are not the Citronella type you may have seen in the past. These are the newer formulated type which have a Synthetic Pyrethrin as the active ingredient - a known repellent to flying insects. Though there may be some which enter the perimeter defense, the Grids or Coils are then there to help prevent them from being able to find their meal! These products are great for around the home and the Grids or Coils are certainly portable enough to bring camping, but how can you repel mosquitoes when you are golfing, fishing or doing other outdoor activities? There are two other types of repellents which can help. The first one is the BUG BAND. This is worn around the wrist and like the Grids, releases a scent which actively confuses mosquitoes and prevents them from identifying you as a target. These will last many hours, can be stored for long term reuse and come in both adult and children sizes. The latest of the personal repellers which works well is the PERSONAL BITE SHIELD. This unit is made of a strong durable plastic and features a small fan which is powered by two AA batteries. The unit holds repellent cartridges which have the latest mosquito repellent, Geraniol, which is all natural. Geraniol is extracted from plants where it serves as a natural repellent plants use to keep predatory insects away. Each cartridge is snapped onto the Shield and the fan is turned on which disperses the Geraniol around the person wearing the device. It's about the size of a pager, fits on your belt or can be set on a table, ledge or other area where you are sitting. The pleasant light odor released will keep mosquitoes at bay naturally and it can even be hooked up to an external power supply. If you want something for your skin, which is the most direct way of repelling mosquitoes, consider either CITRONELLA LOTION or DEET. Both will repel mosquitoes as well as other biting insects. Citronella is not nearly as strong as the Deet and expect to apply it several times a day. Deet will last much longer and do a better job overall at keeping most any type of pest away. It comes in different strengths and flavors with the 100% form being a low odor mix. For complete control when traveling abroad or into areas where mosquitoes pose a real health risk, use some PERMETHRIN to treat clothing, gear and shoes. Permethrin is odorless, labeled for many uses and will keep mosquitoes away since it acts as a detectable repellent they don't like. It is important to understand that the products listed above are not "controlling" local infestations. They should be used when other methods of true control are not an option or if you only want some temporary relief. Repellents of today are better than ever; however, they do just that - repel. You may want to keep a jar of MOSQUITO OINTMENT handy for when you end up getting bit. This soothing treatment takes the itch away and promotes fast healing. Small enough to take out into the field and since only a little is needed, it goes a long way. Another option is to keep them off you all together when going afield. This can be done with our MOSQUITO HEAD NETS. These are lightweight and come with elastic bands. Simply pull them over your head and they will fit snug enough to keep mosquitoes off your face but loose enough so you will still be comfortable. These are an excellent item to bring along when fishing or camping and the pressure from mosquitoes or other biting pests is simply too great. The screen is pre-treated with Permethrin so it offers some repellency as well. If you want no chemicals on it simply wash it once and all the Permethrin will be gone. However, we suggest spraying it periodically with our Permethrin Aerosol featured above. This treatment will help to keep them away from you all together adding that extra protection sometimes needed. We also have four types of netting which can be used for sleeping. These include the TRACKER NETTING, the SINGLE BED NETTING, the DOUBLE BED NETTING and the CANOPY NETTING. All are made from the same light screening which lets air flow unrestricted but is sure to keep out any biting insect. All come with rope allowing you to hang them from a tree or ceiling; all are pre-treated with Permethrin. The Tracker is designed for campers. It comes with it's own storage pouch making it easy to carry along in any backpack. It won't take up a lot of space but it will certainly make the trip a lot more enjoyable. The Single Bed Netting is for just that - a single bed. Use it where windows are kept open inviting night time biting insects. The Double Bed Netting is the same look and design. It's just large enough for a double bed. The Canopy Bed Netting is the more elaborate and complete design which can be used to elegantly dress up any bed yet provide protection from flying pests. It features an entrance which overlaps which is fashionable as well as usable. Though the Tracker is best suited to go afield, any of them can be brought with you as you travel. The Single and Double Bed Netting come in a simple heavy plastic bag in which to store them when not in use; the Canopy Bed Netting comes in a white burlap bag. MOSQUITO DUNKS look like a donut and are used in water where mosquitoes are reproducing. The dunk will slowly melt away releasing thousands of bacteria which will kill mosquito larva. By killing the larva, the mosquito reproduction will stop. This has long been the approach people have used when controlling mosquito populations. The dunks are so safe they can be used in ponds, bird baths and water holding tanks without being a hazard to pets, wildlife or people. They can be used in retention ponds, catch basins for plants and drainage ditches. Use them anywhere you know that water will be held for 3 weeks or more. Because the bacteria is simply digested by mammals (which includes pets, people and wildlife), there is no hazard to this product being used in water used for drinking. If you have water which will only be available for 1-3 weeks, you can still treat with Mosquito Dunks but MOSQUITO GRANULES will probably work better. Because they release quicker, they will impact the developing mosquitoes that much faster. Mosquito Granules have a short life; you may need to treat once a week since they break down so fast. However, they are perfect for small areas such as plant catch pans, bird baths and rain barrels. Made from the same bacteria as the original dunk, Mosquito Granules are able to stop developing mosquitoes from reaching adulthood. If you want something like the Mosquito Granules but a little stronger, get the METHOPRENE GRANULES. These look like the other granules but use a growth regulator instead of just a bacteria to impact growing mosquitoes. The active ingredient in this granule is Methoprene. Long used for flea control, Methoprene will stop mosquitoes from being able to mature to reproducing adults. Since Methoprene is essentially a protein, this product is still very safe to use and is labeled for all the same areas as the other granules. However, there is no hit or miss with this product. Any mosquito larva that are in treated water won't be able to grow right or fully mature so this is great to use in ditches, free standing water, moist and wet compost or flower beds, water gardens, tree holes, roof gutters, pool covers and just about anywhere water is able to pool. The other great thing about this form is that a little goes a long way making it much more practical to use. You will have to apply it at least once a month but regular applications will prevent local activity from completing their life cycle. If you have a lot of plant life and landscape around the home that requires water throughout the growing season, chances are you will attract mosquitoes. Shrubs, annual flowers, thick Fescue and Bermuda grass or ivy all provide pockets of moisture where water can last and provide shelter for mosquitoes. As warm and hot summer months dry local wet lands, expect mosquitoes to migrate in search of moist, shady areas. Many pool owners or homes with decks and porches provide perfect conditions for mosquitoes. The shade and moist areas are what mosquitoes need to survive and you will notice populations increasing as it the hot summer sun diminishes local watering holes. Use a residual product to treat these areas. ESFENVALERATE is a great product to use when treating these areas. It is easy to apply, is labeled for use on turf, trees, shrubs and flowers and will provide residual for long term control. Mosquitoes are easy to kill. Esfenvalerate will last several weeks on treated surfaces and does very well under decks, porches and other shady areas mosquitoes are attracted to. Use a DIAL-A-MIX applicator which hooks to your garden hose to apply Esfenvalerate as it requires a lot of water. It is used at a low rate so a little bit goes a long way. You will see immediate results following the initial treatment. It is hard to say how long you can expect to be mosquito free. Every area will experience different results based on weather, moisture, wind, local breeding conditions, rain, temperature and humidity. Expect to get 1-2 weeks of protection per treatment. Dry environments can expect longer lengths of residual. Retreat as necessary. If you are going to be doing some residual spraying with the Esfenvalerate, a good idea is to add a growth regulator to the mix. This will enable you to get long term control over the areas being treated. Long used for flea control, growth regulators have now found a niche for controlling mosquitoes. Earlier in this article, the Methoprene Granules were highlighted. They use a growth regulator and are designed to be applied in water. Another growth regulator that is designed to be sprayed out is NYLAR. This product is newer then Methoprene but works much the same way. Basically it interferes with the mosquito larva's ability to grow properly so they are not able to fully develop to biting adults. Use it with the Esfenvalerate since Nylar won't kill any adults; it only works on eggs and larva stages. Though you can use a standard pump sprayer to apply the two materials, if you have larger areas to treat, the Dial-A-Mix Sprayer featured above will better serve you. Just add 1 oz of the Nylar with 1 oz of the Esfenvalerate for every 1500 sq/ft of area you want to treat and set the dial to 4 for a good spray mix. Adding the Nylar to the tank mix really makes sense and will cut down on the frequency of treatments needed by about 75%. In most cases, Esfenvalerate by itself will have to sprayed at least twice a month. When used with the Nylar, you will only have to treat once every 2 months. Combining the two products will cost more at first but in the long run will end up saving you a lot of work, a lot of time and a lot of cost. Space spraying has long been the standard way to treat for mosquitoes and may be necessary in certain areas. Since mosquitoes are able to fly, they may be coming to your yard for food and living directly alongside your land in wet, swampy conditions that offer shade and moisture. Such conditions, when large, are hard to treat with Esfenvalerate as described above. To penetrate dense foliage or to cover large areas quickly, space spraying is the preferred method of treatment. You may be able to treat small areas like this with an aerosol like PT-565XLO. This product uses pyrethrum as the active ingredient and will kill mosquitoes quickly. Spray it around decks and picnic areas to provide yourself with a few hours of relief. It is easy to use and is great for around the garden, deck, patio or back yard area. AEROSOL MACHINES are also good for small areas. They can be hung on the wall, are battery operated and can be programmed to release a blast of pyrethrin based aerosol which works well on flying insects. Use some METERED AEROSOL in these machines and they will keep most any flying pest under control. Use of these products is for small areas only. If you are treating a larger area, use an electrical fogging machine such as a FM6309. These machines are able to pump a lot of chemical over a large area in a short amount of time. They create a "fog" or "mist" which floats around plant leaves and other areas mosquitoes like to hide. This machine produces a cold fog which is adequate for most home owner applications. It is able to treat 1/4 to 1/2 acre in less than 30 minutes. (The machine will pump a gallon of material in around 12 minutes, but it takes extra time to drag the extension cord to new treatment areas.) If you have a lot of small areas to treat, you may prefer the FM6208. This fogger is a step up from the FM6309 because it has a volume control switch which allows you to adjust the rate of flow. It still uses electricity to power it, but unlike the FM6309 it has a rotation switch which can be set to off, low, medium or high. This can be a real help for two reasons. First, you are able to turn it keep it low when treating around the home and you only need a light mist. Such treatments can be tough with the FM6309 because it only fogs at one speed - high! When you try to treat alongside a structure, the fog is so strong it will bounce off the building and come back at you. If you have to do a lot of this type of fogging, get the FM6208. It not only lets you turn it down for these types of delicate applications but the rotating switch will let you turn it to off. Though the machine is still on and trying to fog, it will quickly run dry. This is handy if you are interrupted during treatment. Simply turn the switch to off and let the fogger use up what's in it's system. Now you can store it for a few days without worrying about the insides getting clogged. Two other models you may want to consider are the FM7807 and the MINI FOGGER. The FM7807 features the same controls as the FM6208 but also has a long extension. This extension is great for reaching high, under and around things which other machines have difficulty covering. The FM7807 also features a quick on/off control on the wand; great for when you need precise control of flow. The Mini Fogger is basically a compact version designed for the person who has only a very small area to treat. It can cover areas from 2500-5000 sq/ft very well. It uses electricity, has a volume control and a quart holding tank. Great for the condo or townhouse owner who has a small area to treat. All these foggers are great for jobs around the home but they do require electricity and are not nearly as portable as the BACKPACK FOGGER. This device has over a two gallon tank and is gas powered. Perfect for those large jobs where electricity is not available. Simply fill up the holding tank, make sure you have enough gas in the tank and you can go wherever the mosquitoes may be hiding! Great for property where being free from electric cords is necessary or if you have a lot of areas to treat on a regular basis but need to move from one point to another as you treat. Once you decide on which type of fogger will fits your needs, you will have to get some chemical. Use water as the carrier for the tank mix and try to reach as much as the shady, undercover where mosquitoes tend to hide. Though products like Malathion have been used over the years for such applications, you will achieve better results with Pyrethrum and Permethrin and they have little if any odor. PYRETHRUM is natures own pesticide and is very active on mosquitoes. It will kill adults on contact and works on many other flying insects as well. As good as Pyrethrum is, it will not provide any residual. Mix PERMETRHIN in the tank for better results. Permethrin does not provide the quick kill like Pyrethrum but it does last from 1-2 weeks. This will prove helpful when treating large areas. The residual will help to stop the mosquitoes from reproducing or from providing shelter to wanderers that like the moist shady shelter a large wooded area adjacent to your home may provide. The one-two punch of pyrethrum and permethrin will kill existing populations of mosquitoes in hard to treat vegetation and keep new infestations from being able to form. And though this combination is hard to beat, the addition of some NYLAR to the mix could help cut down on the frequency of treatments needed throughout the season. As explained above, Nylar works on eggs and larval stages of mosquitoes and by doing so will prevent their young from reaching maturity. This "breaking of the "cycle" means that fewer treatments will be needed over the course of the season and by having this impact, the amount of time and chemical used should be decreased as well. If you have larger areas to fog than an acre, you should consider moving up to a thermal fogger. Thermal foggers are "hot"; that is, they heat the chemical before it is released. This type of fog is lighter and comprised of smaller sized particles than that of a cold fog. Thermal foggers are more thorough during application and require an oil based formulation instead of water. The fog will stay in the air longer and penetrate more providing better performance than fogs created by electric machines. However, there are some tradeoff for this better performance. First, machines are costly. Don't waste you time with $100.00 to $200.00 units found at some hardware stores. These devices will create some fog, but the flow is so weak and slow you are better off using the PT-565XLO listed above. If you have anything larger than a few thousand feet to treat, these cheap machines are just not able to do the job adequately. If you have a large area that you want to keep mosquito free, invest your money with a machine that will do the job. The GE THERMAL FOGGER is gas powered, electric start and lightweight. It can be carried easily to treat large areas or you can let it run out the back of your ATV or truck as you drive through the property you are treating. This machine will create a fog which is light, penetrating and very effective for mosquito control. Be sure to use the proper material when fogging to achieve the best results. Oil based PYRETHRUM or SYNTHETIC PYRETHRUM are needed. These formulations will provide quick knockdown of existing populations and you can add some Permethrin to the mix as detailed above to get extra residual. If you intend on doing a lot of fogging, you should consider getting our DEODORIZED OIL in bulk form and then adding Pyrethrum and Permethrin to the mix. This will prove to be the most economical formulation available when treating large areas many times during the season. Like fogging with electric foggers you may need to treat twice a week to get control of bad problem areas, but once you knock down the local population you will probably only have to treat once every week or two. Another option that needs to be mentioned is the installation of a Mosquito Killing Machine. There are several being made these days and most any retailer has one style or another. These machines all use some type of attractant to lure mosquitoes to them. It could be CO2, heat, moisture or something else that biting insects find attractive. Once lured to the device, most have a mechanism that will somehow either kill or remove the pest. The goal of these machines is to remove as many pests as possible in your yard. Though most will kill a lot of insects, it is not likely that any will adequately remove enough to have the desired result. We have carried many different such devices over the years and though several have proven to do a good job of killing mosquitoes, none have proven to keep a designated outside area mosquito free. Since these applications only kill adults and do nothing to address the reproduction sites, larval stages and other key components of the mosquitoes life cycle, they don't have nearly enough of an impact to warrant implementation. Since most people purchase these units with the hopes that they won't get bit anymore, we have decided to drop all but one model at this time. The model we continue to carry is mostly designed for inside applications but can be used for small decks, enclosed patios or screened in rooms. Such areas usually only have a few flying insect problems and this machine will do a good job of killing off any biting pests which may be around. For larger areas, use one of our foggers or liquid materials for the best control. But if you have a room or two in the home which seems to get a lot of mosquitoes or other biting pests, install one of our INDOOR MOSQUITO TRAPS. It has several attractants that do a good job of attracting local mosquitoes and can handle small confined areas well. Don't get it with the hopes that you will never see another mosquito or biting pest; such results are not likely. However, they will help and general rule when treating for mosquitoes is that the more materials being used, the better. Another type of trap which is relatively new but will prove to be quite helpful when dealing with mosquitoes is called the MOSQUITO PHEROMONE TRAP. This trap is made of a small plastic jar which is about 1/2 gallon in size. The jar is filled with one quart of water and some "vegetation tablets". These tablets are basically plants which have been freeze dried and once mixed with water will convert back to their old decaying natural state. Such areas female mosquitoes will seek out as good egg laying locations.Be sure to used distilled water or let a quart of water sit overnight so the chlorine level dissipates. Chlorine will disrupt the natural decaying process and prevent certain odors from releasing. This will interfere with the mosquitoes ability to find the trap. Add the water to the jar along with the pellets and then about 3 drops of dish detergent. The jar has a small tube that runs through its middle which serves as an entrance as well as a holding area for a EGG LAYING PHEROMONE which is then set out with the trap. Be careful not to touch the pheromone; human scent, natural oil from our fingers or some other contaminate will erode its effectiveness. The pheromone is a scent which mosquitoes naturally release in the wild when they find a good egg laying location. This "locator pheromone" is then smelled by local mosquitoes ready to lay eggs and will attract them inside. Once inside, they will attempt to land on the water. At that point, the detergent which was added to the tank will cause them to sink into the water and drown! This will happen over and over during the course of the season which can have an astounding impact on local populations. Try to place out 2-4 traps for the average yard; about 6 per 1/2 acre. The pheromone should be replaced every month and be sure the water level does not drop too low during the hot and dry season. Also, be sure to keep Traps along property borders or where people won't be active; preferably in moist shady areas. You can also equip yourself with a HAND HELD ZAPPER. This is really handy when dealing with mosquitoes which are sometimes hard to swat. Simply wave the Zapper where the mosquito is flying in a gentle manner and once it makes contact with the Zappers grid it will be dead. Don't try to swat them, this Zapper is not designed for such use. However, it is a great tool to keep at your side when mosquitoes are in the area and you want an easy and clean way to kill them. And since mosquito's are so slow when flying, you can even catch them with our BUG VACUUM/ZAPPER cordless tool. This device is generally best for crawling insects but works well for mosquitoes and just about any flying pest which likes to land on walls, windows or ceilings. Though effective at killing many mosquitoes, don't be lured into using these devices because it is so easy to install and operate. It is another tool and when dealing with mosquitoes, the best approach is to incorporate as many tools as is needed. You will get much better results if you treat property with Esfenvalerate or Fog and install a Mosquito Killer. The final method of keeping mosquitoes away can be done with a Mosquito Blocker. This is relatively new technology but the logic behind it makes a lot of sense. It's kind of like the new allergy medicines. Most allergy medicine is designed to deal with the symptoms of the allergy - the itchy eyes, running nose and congestion. Newer medications are histamine blockers which essentially block the uncomfortable symptoms of having allergies. You still have the allergy - just not the symptom. This approach is very effective, relatively easy to administer and tends to be healthier in the long run. The same is true for the Mosquito Blockers. These devices are small plastic machines which run on 2 AA batteries. They will operate at least 720 hours and are used to power a small fan. The fan blows out a product called Conceal. This is not a pesticide. Conceal is comprised of plant oils and other natural ingredients which bind to insect olfactory sensors. The impact of Conceal is that mosquitoes and several other biting insects are not able to detect Co2 or octenol - two gases which people (mammals) exhale all the time. By not being able to detect the presence of Co2 or octenol mosquitoes further than 30 feet away will not be able to track your presence. You will become invisible to them. Remember, mosquitoes have a limited range of sight. It is probably not more than 15-20 feet and certainly not more than 30 feet. Mosquitoes which are not able to see any targets will then begin to trail or track their prey by detecting Co2 and octenol. As they fly around they are constantly hoping to find some of these gases in the air. Any breeze will carry your exhaled breath several hundred feet and mosquitoes along this path will be able to trace it back to it's source - YOU! By installing a MOSQUITO BLOCKER about 10-20 feet downwind of where you are sitting, the Conceal will be carried with your exhaled gases. As mosquitoes cross this path they will be affected by the Conceal in such a way that they will not be able to detect the Co2 or octenol. These units work best when there is a slight breeze. Simply place a unit close by, downwind, and go about your activities. You won't even know it is there after a few days. It's perfect for the front porch, decks, sitting areas out in the yard, pool or pond and is completely portable. Just remember to bring it back inside and to keep a fresh supply of CONCEAL in it to enable to work properly. If there is no wind or breeze present, you will probably need to locate two or three machines around you. Remember, the more the better. If there is no real direction to the way the air is moving then try to keep one on any side mosquitoes can approach. This may mean having 2,3 or even 4 machines. As a general rule you need to have more machines as the number of people increases as well. This is due to the increase of gases being released. The Mosquito Blockers are an excellent tool which can be set out easily and will help provide additional relief from the pressure local mosquitoes will apply. When used in conjunction with Mosquito Killers or Foggers you can really reduce the amount of bites you, family and friends have to endure. Mosquitoes have long been an enemy of man and our lifestyle. To enjoy the great outdoors, you must first learn why they are attracted to your land. Once shelter and breeding locations have been identified, choose the best approach to stopping them. Though the Bug Bands and Deet will help if you are out in the field, treatments with our Repellent Granules, Dunks, Esfenvalerate or Fogging Compounds will be necessary for complete and pest free areas around the home. Be sure to get enough "machine" when fogging so the time you spend treating will be minimal and the time you are able to enjoy the great outdoors is maximized. Set out some Blockers for added protection and you just might be able to enjoy the great outdoors once again! To see any of the products listed above, click on their name as it appears in a different color or underlined. The link will bring you to our product catalog where you will be able to see the product and learn more about it. You can also link to our product catalog below. Check out the rest of our catalog and make sure to keep us bookmarked! We've got the products for whatever is bugging you! CLICK HERE TO GO TO OUR MOSQUITO PRODUCTS CLICK HERE TO GO TO OUR PRODUCT SELECTION PAGE CLICK HERE TO GO BACK TO OUR ARTICLE SELECTION PAGE CLICK HERE TO GO TO OUR MAIN PAGE Our toll free number is 1-800-877-7290. E-Mail us at support@bugspray.com All articles copy righted by U-Spray, Inc. 4653 Highway 78 Lilburn, Georgia 30047 Phone: (770)985-9388 Fax: (770)985-9319 Toll Free: 1-800-877-7290 url: http://www.bugspray.com/articles98/mosquitoes.html

HOME REMEDIES, HOLISTIC APPROACH, & REPELLENT PLANTS - Chinaroad Lowchens of Australia

2005-10-11T20:20:00.000-07:00

HOME REMEDIES, HOLISTIC APPROACH, & REPELLENT PLANTS - Chinaroad Lowchens of Australia

Some suggested old home remedies. No endorsement is intended nor liability assumed since most of these home remedies are not proven or approved as pest control recommendations.

1.

Banish ants from your pet's food dish by wiping the floor under and around it with a cloth dipped in kerosene. Then stand the food dish in a larger dish containing water.
2.

Keep ants from crawling up a picnic table by standing each leg in a small pan of water.
3.

To kill ants, use a paste of equal parts of borax and confectioner sugar.
4.

Mix mint apple jelly and boric acid for ant control (two tablespoons boric acid powder per 10 ounces of mint apple jelly).
5.

Leave a few tea bags of mint tea near areas where the ants seem most active. Dry, crushed mint leaves or cloves also work as ant deterrents.
6.

Keep a small spray bottle handy, and spray the ants with a bit of soapy water.
7.

Mix peanut butter (six parts), brown sugar (one part), one-half teaspoon salt with boric acid (one part) for ant control.
8.

For ant control, spray vinegar around door and window frame, under appliances, and along other known ant trails.
9.

If ants are coming in through doors or windows, put a cinnamon stick across the path. They will not cross it.
10.

Mix three cups water, one cup sugar and four teaspoons boric acid powder for ant control. (Pour a over a cotton wad in a small dish or bottle cap.)
11.

Sliced or crushed cucumbers to keep cockroaches away from food.
12.

Mix equal parts of boric acid powder, powdered sugar, and cornmeal as a poison bait for cockroaches.
13.

Mix equal parts of plaster of Paris and powdered sugar as a poison bait for cockroaches.
14.

It is a little known fact that roaches like high places. If you put borax on TOP of your kitchen cabinets (not inside), if space allows between ceiling and cabinets, the roaches will take the borax to their nests, killing all of them.
15.

Keep a spray bottle of soapy water on hand. Spraying roaches directly with soapy water will kill them.
16.

Walk through a room wearing white socks to detect fleas. Dark fleas jumping on the white background are easily seen.
17.

Use banana peels to repel fleas.
18.

Feed yeast to dogs to repel fleas.
19.

Fleas HATE Stash Earl Grey. Tear open a few bags, scatter the tea about on your carpet and vacuum up in a few days.
20.

For flea control, add a little vinegar to your pet's drinking water to fight fleas and mange.
21.

For flea control add ½ teaspoon to the wash water or a few drops to the pets shampoo.
22.

Suspend a light bulb over a pan of oil or soapy water to attract and drown fleas during the night.
23.

For a fly repellent - 2 cups vinegar, 1 cup Avon Skin So Soft, 1 cup water, 1 tablespoon Eucalyptus oil, 1 tablespoon citronella oil - Put in a spray bottle and spray dog's coat.
24.

Mix water with cornstarch into a paste and apply. This is effective in drawing out the poisons of most insect bites and is also an effective remedy for diaper rash.
25.

Rub jewel weed on mosquito bites and poison ivy to control itch.
26.

For mosquito bites apply lime juice diluted with water on bites with cotton ball.
27.

Mozzies won't bite if you mix 4 parts glycerine, 4 parts alcohol, 1 part eucalyptus oil. Or make a solution of equal parts of isopropyl alcohol and methyl phthalate.
28.

If you're using the barbeque, throw a bit of sage or rosemary on the coals to repel mosquitos.
29.

Put an opened bottle of Oil of Pennyroyal Essence in the room you want mosquito free.
30.

Use Avon's Skin-So-Soft as an insect repellent for people and pets (good mosquito repellent). It helps relieve itching caused by insect bites and dry skin. Also, mix five parts water, one part Skin-So-Soft and mist on show animals. Brush in to make their coats gleam and keep insects off so your animal won't fidget.
31.

Use hedge apples for control of crickets and spiders.
32.

For grass and weeds growing between stones or bricks on walks or terraces, sprinkle 20 Mule Team borax powder and sweep into cracks (one application every other year).
33.

Apply tobacco and snuff juice for wasp stings and bites.
34.

Use beer or yeast dissolved in water in pit fall traps (cups sunk into the ground) to attract and drown snails and slugs.
35.

Prevent mosquitoes from breeding in rain barrels by applying 1 tablespoon of olive oil to the water's surface.
36.

Tick and Fly Spray - two cups white vinegar, one cup Avon Skin-So-Soft bath oil, one cup water, one tablespoon eucalyptus oil (available at drugstores and health food stores).
37.

Pour hot boiling water and a strong cleaning detergent down the drain to eliminate nuisance gnats and flies.

PLANTS & HERBS

* Basil - plant close to the house and it will repel mosquitos.
* Lavender - plant around the house to repel flies and mosquitos.
* Castor Bean Plant - plant in pots within the house; replant outdoors to repel mozzies.
* Scented Geraniums - plant in pots and the garden to repel mosquitos
* Citrosa Plants - plant in pots and the garden to repel mosquitos
* Lemon Thyme - plant in pots to repel mosquitos
* Citronella Grass - plant in pots to repel mosquitos
* Common Marigold - plant in pots and the garden to repel mosquitos
* Thai Lemon Grass - (Cymbopogon citratus) is an effective mosquito repellent.
* Rosemary - powdered Rosemary leaves are used as a flea and tick repellent
* Chamomile - repels mosquitos
* Citriodora - repels mosquitos

MORE USEFUL PLANTS FOR YOUR GARDEN

* Red Raspberry Leaves (Rubus idacus) - the leaves strengthen the muscles of the uterus
* Plantain (Plantago lanceolata) - leaves used whole or in powdered form for bleeding cuts
* Hen-and-chicks (houseleek) - Sempervivum tetorum - soothes minor stings and burns
* St. John's Wort (Hypericum perforatum) - used for burns and scrapes
* Yarrow (achillea millefolium) - stops bleeding

OFF THE SHELF LOW COST PEST CONTROL

Introduction to Fluorescent Proteins

2005-10-11T20:15:00.000-07:00

Nikon MicroscopyU: Introduction to Fluorescent Proteins

ntroduction to Fluorescent Proteins

The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology. When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.

Osamu Shimomura and Frank Johnson, working at the Friday Harbor Laboratories of the University of Washington in 1961, first isolated a calcium-dependent bioluminescent protein from the Aequorea victoria jellyfish, which they named aequorin. During the isolation procedure, a second protein was observed that lacked the blue-emitting bioluminescent properties of aequorin, but was able to produce green fluorescence when illuminated with ultraviolet light. Due to this property, the protein was eventually christened with the unceremonious name of green fluorescent protein (GFP). Over the next two decades, researchers determined that aequorin and the green fluorescent protein work together in the light organs of the jellyfish to convert calcium-induced luminescent signals into the green fluorescence characteristic of the species.

Although the gene for green fluorescent protein was first cloned in 1992, the significant potential as a molecular probe was not realized until several years later when fusion products were used to track gene expression in bacteria and nematodes. Since these early studies, green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors that are broadly referred to as fluorescent proteins. More recently, fluorescent proteins from other species have been identified and isolated, resulting in further expansion of the color palette. With the rapid evolution of fluorescent protein technology, the utility of this genetically encoded fluorophore for a wide spectrum of applications beyond the simple tracking of tagged biomolecules in living cells is now becoming fully appreciated.

Illustrated in Figure 1 are two examples of multiple fluorescent protein labeling in living cells using fusion products targeted at sub-cellular (organelle) locations. The opossum kidney cortex proximal tubule epithelial cell (OK line) presented in Figure 1(a) was transfected with a cocktail of fluorescent protein variants fused to peptide signals that mediate transport to either the nucleus (enhanced cyan fluorescent protein; ECFP), the mitochondria (DsRed fluorescent protein; DsRed2FP), or the microtubule network (enhanced green fluorescent protein; EGFP). A similar specimen consisting of human cervical adenocarcinoma epithelial cells (HeLa line) is depicted in Figure 1(b). The HeLa cells were co-transfected with sub-cellular localization vectors fused to enhanced cyan and yellow (EYFP) fluorescent protein coding sequences (Golgi complex and the nucleus, respectively), as well as a variant of the Discosoma striata marine anemone fluorescent protein, DsRed2FP, targeting the mitochondrial network.

Green fluorescent protein, and its mutated allelic forms, blue, cyan, and yellow fluorescent proteins are used to construct fluorescent chimeric proteins that can be expressed in living cells, tissues, and entire organisms, after transfection with the engineered vectors. Red fluorescent proteins have been isolated from other species, including coral reef organisms, and are similarly useful. The fluorescent protein technique avoids the problem of purifying, tagging, and introducing labeled proteins into cells or the task of producing specific antibodies for surface or internal antigens.

Properties and Modifications of Aequorea victoria Green Fluorescent Protein

Among the most important aspects of the green fluorescent protein to appreciate is that the entire 27 kiloDalton native peptide structure is essential to the development and maintenance of its fluorescence. It is remarkable that the principle fluorophore is derived from a triplet of adjacent amino acids: the serine, tyrosine, and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66, and Gly67; see Figure 2). Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. What is unique to the fluorescent protein is that the location of this peptide triplet resides in the center of a remarkably stable barrel structure consisting of 11 beta-sheets folded into a tube.

Within the hydrophobic environment in the center of the green fluorescent protein, a reaction occurs between the carboxyl carbon of Ser65 and the amino nitrogen of Gly67 that results in the formation of an imidazolin-5-one heterocyclic nitrogen ring system (as illustrated in Figure 2). Further oxidation results in conjugation of the imidazoline ring with Tyr66 and maturation of a fluorescent species. It is important to note that the native green fluorescent protein fluorophore exists in two states. A protonated form, the predominant state, has an excitation maximum at 395 nanometers, and a less prevalent, unprotonated form that absorbs at approximately 475 nanometers. Regardless of the excitation wavelength, however, fluorescence emission has a maximum peak wavelength at 507 nanometers, although the peak is broad and not well defined.

Two predominant features of the fluorescent protein fluorophore have important implications for its utility as a probe. First, the photophysical properties of green fluorescent protein as a fluorophore are quite complex and thus, the molecule can accommodate a considerable amount of modification. Many studies have focused on fine-tuning the fluorescence of native green fluorescent protein to provide a broad range of molecular probes, but the more significant and vast potential of employing the protein as a starting material for constructing advanced fluorophores cannot be understated. The second important feature of green fluorescent protein is that fluorescence is highly dependent on the molecular structure surrounding the tripeptide fluorophore.

Denaturation of green fluorescent protein destroys fluorescence, as might be expected, and mutations to residues surrounding the tripeptide fluorophore can dramatically alter the fluorescence properties. The packing of amino acid residues inside the beta barrel is extremely stable, which results in a very high fluorescence quantum yield (up to 80 percent). This tight protein structure also confers resistance to fluorescence variations due to fluctuations in pH, temperature, and denaturants such as urea. The high level of stability is generally altered in a negative manner by mutations in green fluorescent protein that perturb fluorescence, resulting in a reduction of quantum yield and greater environmental sensitivity. Although several of these defects can be overcome by additional mutations, derivative fluorescent proteins are generally more sensitive to the environment than the native species. These limitations should be seriously considered when designing experiments with genetic variants.

In order to adapt fluorescent proteins for use in mammalian systems, several basic modifications of the wild-type green fluorescent protein were undertaken and are now found in all commonly used variants. The first step was to optimize the maturation of fluorescence to a 37-degree Celsius environment. Maturation of the wild-type fluorophore is quite efficient at 28 degrees, but increasing the temperature to 37 degrees substantially reduces overall maturation and results in decreased fluorescence. Mutation of the phenylalanine residue at position 64 (Phe64) to leucine results in improved maturation of fluorescence at 37 degrees, which is at least equivalent to that observed at 28 degrees. This mutation is present in the most popular varieties of fluorescent proteins derived from Aequorea victoria, but is not the only mutation that improves folding at 37 degrees as other variants have been discovered.

In addition to improving the maturation at 37 degrees, the optimization of codon usage for mammalian expression has also improved overall brightness of green fluorescent protein expressed in mammalian cells. In all, over 190 silent mutations have been introduced into the coding sequence to enhance expression in human tissues. A Kozak translation initiation site (containing the nucleotide sequence A/GCCAT) was also introduced by insertion of valine as the second amino acid. These, along with a variety of other improvements (discussed below), have resulted in a very useful probe for live cell imaging of mammalian cells and are common to all of the currently used fluorescent probes derived from the original jellyfish protein.

The Fluorescent Protein Color Palette

A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1). Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.
Fluorescent Protein Properties
Protein
(Acronym) Excitation
Maximum
(nm) Emission
Maximum
(nm) Molar
Extinction
Coefficient Quantum
Yield in vivo
Structure Relative
Brightness
(% of EGFP)
GFP (wt) 395/475 509 21,000 0.77 Monomer 48
Green Fluorescent Proteins
EGFP 484 510 56,000 0.60 Monomer 100
Emerald 487 509 57,500 0.68 Monomer 116
Azami Green 492 505 55,000 0.74 Monomer 121
CopGFP 482 502 70,000 0.60 Monomer 125
AcGFP 480 505 50,000 0.55 Monomer 82
ZsGreen 493 505 43,000 0.91 Tetramer 117
Blue Fluorescent Proteins
EBFP 383 445 29,000 0.31 Monomer 27
Sapphire 399 511 29,000 0.64 Monomer 55
T-Sapphire 399 511 44,000 0.60 Monomer 79
Cyan Fluorescent Proteins
AmCyan1 458 489 44,000 0.24 Tetramer 31
ECFP 439 476 32,500 0.40 Monomer 39
Cerulean 433 475 43,000 0.62 Monomer 79
Midoriishi Cyan 472 495 27,300 0.90 Dimer 73
Yellow Fluorescent Proteins
EYFP 514 527 83,400 0.61 Monomer 151
PhiYFP 525 537 130,000 0.40 Monomer 155
Citrine 516 529 77,000 0.76 Monomer 174
Venus 515 528 92,200 0.57 Monomer 156
ZsYellow1 529 539 20,200 0.42 Tetramer 25
Orange and Red Fluorescent Proteins
Kusabira Orange 548 559 51,600 0.60 Monomer 92
mOrange 548 562 71,000 0.69 Monomer 146
DsRed 558 583 75,000 0.79 Tetramer 176
DsRed2 563 582 43,800 0.55 Tetramer 72
DsRed-Express 555 584 38,000 0.51 Tetramer 58
mTangerine 568 585 38,000 0.30 Monomer 34
mStrawberry 574 596 90,000 0.29 Monomer 78
AsRed2 576 592 56,200 0.05 Tetramer 8
mRFP1 584 607 50,000 0.25 Monomer 37
mCherry 587 610 72,000 0.22 Monomer 47
HcRed1 588 618 20,000 0.015 Dimer 1
mRaspberry 598 625 86,000 0.15 Monomer 38
HcRed-Tandem 590 637 160,000 0.04 Monomer 19
mPlum 590 649 41,000 0.10 Monomer 12

Table 1

Presented in Table 1 is a compilation of properties displayed by several of the most popular and useful fluorescent protein variants. Along with the common name and/or acronym for each fluorescent protein, the peak absorption and emission wavelengths (given in nanometers), molar extinction coefficient, quantum yield, relative brightness, and in vivo structural associations are listed. The computed brightness values were derived from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP. This listing was created from scientific and commercial literature resources and is not intended to be comprehensive, but instead represents fluorescent protein derivatives that have received considerable attention in the literature and may prove valuable in research efforts. Furthermore, the absorption and fluorescence emission spectra listed in tables and illustrated below were recorded under controlled conditions and are normalized for comparison and display purposes only. In actual fluorescence microscopy investigations, spectral profiles and wavelength maxima may vary due to environmental effects, such as pH, ionic concentration, and solvent polarity, as well as fluctuations in localized probe concentration. Therefore, the listed extinction coefficients and quantum yields may differ from those actually observed under experimental conditions.

Green Fluorescent Proteins

Although native green fluorescent protein produces significant fluorescence and is extremely stable, the excitation maximum is close to the ultraviolet range. Because ultraviolet light requires special optical considerations and can damage living cells, it is generally not well suited for live cell imaging with optical microscopy. Fortunately, the excitation maximum of green fluorescent protein is readily shifted to 488 nanometers (in the cyan region) by introducing a single point mutation altering the serine at position 65 into a threonine residue (S65T). This mutation is featured in the most popular variant of green fluorescent protein, termed enhanced GFP (EGFP), which is commercially available in a wide range of vectors offered by BD Biosciences Clontech, one of the leaders in fluorescent protein technology. Furthermore, the enhanced version can be imaged using commonly available filter sets designed for fluorescein and is among the brightest of the currently available fluorescent proteins. These features have rendered enhanced green fluorescent protein one of the most popular probes and the best choice for most single-label fluorescent protein experiments. The only drawbacks to the use of EGFP are a slight sensitivity to pH and a weak tendency to dimerize.

In addition to enhanced green fluorescent protein, several other variants are currently being used in live-cell imaging. One of the best of these in terms of photostability and brightness may be the Emerald variant, but lack of a commercial source has limited its use. Several sources provide humanized green fluorescent protein variants that offer distinct advantages for fluorescence resonance energy transfer (FRET) experiments. Substitution of the phenylalanine residue at position 64 for leucine (F64L; GFP2) yields a mutant that retains the 400-nanometer excitation peak and can be coupled as an effective partner for enhanced yellow fluorescent protein. A variant of the S65C mutation (normally substituting cysteine for serine) having a peak excitation at 474 nanometers has been introduced commercially as a more suitable FRET partner for enhanced blue fluorescent protein than the red-shifted enhanced green version. Finally, a reef coral protein, termed ZsGreen1 and having an emission peak at 505 nanometers, has been introduced as a substitute for enhanced green fluorescent protein. When expressed in mammalian cells, ZsGreen1 is very bright relative to EGFP, but has limited utility in producing fusion mutants and, similar to other reef coral proteins, has a tendency to form tetramers.

Yellow Fluorescent Proteins

The family of yellow fluorescent proteins was initiated after the crystal structure of green fluorescent protein revealed that threonine residue 203 (Thr203) was near the chromophore. Mutation of this residue to tyrosine was introduced to stabilize the excited state dipole moment of the chromophore and resulted in a 20-nanometer shift to longer wavelengths for both the excitation and emission spectra. Further refinements led to the development of the enhanced yellow fluorescent protein (EYFP), which is one of the brightest and most widely used fluorescent proteins. The brightness and fluorescence emission spectrum of enhanced yellow fluorescent protein combine to make this probe an excellent candidate for multicolor imaging experiments in fluorescence microscopy. Enhanced yellow fluorescent protein is also useful for energy transfer experiments when paired with enhanced cyan fluorescent protein (ECFP) or GFP2. However, yellow fluorescent protein presents some problems in that it is very sensitive to acidic pH and loses approximately 50 percent of its fluorescence at pH 6.5. In addition, EYFP has also been demonstrated to be sensitive to chloride ions and photobleaches much more readily than the green fluorescent proteins.

Continued development of fluorescent protein architecture for yellow emission has solved several of the problems with the yellow probes. The Citrine variant of yellow fluorescent protein is very bright relative to EYFP and has been demonstrated to be much more resistant to photobleaching, acidic pH, and other environmental effects. Another derivative, named Venus, is the fastest maturing and one of the brightest yellow variants developed to date. The coral reef protein, ZsYellow1, originally cloned from a Zoanthus species native to the Indian and Pacific oceans, produces true yellow emission and is ideal for multicolor applications. Like ZsGreen1, this derivative is not as useful for creating fusions as EYFP and has a tendency to form tetramers. Many of the more robust yellow fluorescent protein variants have been important for quantitative results in FRET studies and are potentially useful for other investigations as well.

Illustrated in Figure 3 are the absorption and emission spectral profiles for many of the commonly used and commercially available fluorescent proteins, which span the visible spectrum from cyan to far red. The variants derived from Aequorea victoria jellyfish, including enhanced cyan, green, and yellow fluorescent proteins, exhibit peak emission wavelengths ranging from 425 to 525 nanometers. Fluorescent proteins derived from coral reefs, DsRed2 and HcRed1 (discussed below), emit longer wavelengths but suffer from oligomerization artifacts in mammalian cells.

Blue and Cyan Fluorescent Proteins

The blue and cyan variants of green fluorescent protein resulted from direct modification of the tyrosine residue at position 66 (Tyr66) in the native fluorophore (see Figure 2). Conversion of this amino acid to histidine results in blue emission having a wavelength maxima at 450 nanometers, whereas conversion to tryptamine results in a major fluorescence peak around 480 nanometers along with a shoulder that peaks around 500 nanometers. Both probes are only weakly fluorescent and require secondary mutations to increase folding efficiency and overall brightness. Even with modifications, the enhanced versions in this class of fluorescent protein (EBFP and ECFP) are only about 25 to 40 percent as bright as enhanced green fluorescent protein. In addition, excitation of blue and cyan fluorescent proteins is most efficient in spectral regions that are not commonly used, so specialized filter sets and laser sources are required.

Despite the drawbacks of blue and cyan fluorescent proteins, the widespread interest in multicolor labeling and FRET has popularized their application in a number of investigations. This is especially true for enhanced cyan fluorescent protein, which can be excited off-peak by an argon-ion laser (using the 457-nanometer spectral line) and is significantly more resistant to photobleaching than the blue derivative. In contrast to other fluorescent proteins, there has not been a high level of interest for designing better probes in the blue region of the visible light spectrum, and a majority of the developmental research on fluorophores in this class has been focused on cyan variants.

Among the improved cyan fluorescent proteins that have been introduced, AmCyan1 and an enhanced cyan variant termed Cerulean show the most promise. Derived from the reef coral, Anemonia majano, the AmCyan1 fluorescent protein variant has been optimized with human codons to generate a high relative brightness level and resistance to photobleaching when compared to enhanced cyan fluorescent protein during mammalian expression. On the downside, similar to most of the other reef coral proteins, this probe has a tendency to form tetramers. The Cerulean fluorescent probe was developed by site-directed mutagenesis of enhanced cyan fluorescent protein to yield a higher extinction coefficient and improved quantum yield. Cerulean is at least 2-fold brighter than enhanced cyan fluorescent protein and has been demonstrated to significantly increase the signal-to-noise ratio when coupled with yellow-emitting fluorescent proteins, such as Venus (see Figure 4), in FRET investigations.

Red Fluorescent Proteins

A major goal of fluorescent protein development has become the construction of a red-emitting derivative that equals or exceeds the advanced properties of enhanced green fluorescent protein. Among the advantages of a suitable red fluorescent protein are the potential compatibility with existing confocal and widefield microscopes (and their filter sets), along with an increased capacity to image entire animals, which are significantly more transparent to red light. Because the construction of red-shifted mutants from the Aequorea victoria jellyfish green fluorescent protein beyond the yellow spectral region has proven largely unsuccessful, investigators have turned their search to the tropical reef corals.

The first coral-derived fluorescent protein to be extensively utilized was derived from Discosoma striata and is commonly referred to as DsRed. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at 583 nanometers whereas the excitation spectrum has a major peak at 558 nanometers and a minor peak around 500 nanometers. Several problems are associated with using DsRed, however. Maturation of DsRed fluorescence occurs slowly and proceeds through a time period when fluorescence emission is in the green region. Termed the green state, this artifact has proven problematic for multiple labeling experiments with other green fluorescent proteins because of the spectral overlap. Furthermore, DsRed is an obligate tetramer and can form large protein aggregates in living cells. Although these features are inconsequential for the use of DsRed as a reporter of gene expression, the usefulness of DsRed as an epitope tag is severely limited. In contrast to the jellyfish fluorescent proteins, which have been successfully used to tag hundreds of proteins, DsRed conjugates have proven much less successful and are often toxic.

A few of the problems with DsRed fluorescent proteins have been overcome through mutagenesis. The second-generation DsRed, known as DsRed2, contains several mutations at the peptide amino terminus that prevent formation of protein aggregates and reduce toxicity. In addition, the fluorophore maturation time is reduced with these modifications. The DsRed2 protein still forms a tetramer, but it is more compatible with green fluorescent proteins in multiple labeling experiments due to the quicker maturation. Further reductions in maturation time have been realized with the third generation of DsRed mutants, which also display an increased brightness level in terms of peak cellular fluorescence. Red fluorescence emission from DsRed-Express can be observed within an hour after expression, as compared to approximately six hours for DsRed2 and 11 hours for DsRed. A yeast-optimized variant, termed RedStar, has been developed that also has an improved maturation rate and increased brightness. The presence of a green state in DsRed-Express and RedStar is not apparent, rendering these fluorescent proteins the best choice in the orange-red spectral region for multiple labeling experiments. Because these probes remain obligate tetramers, they are not the best choice for labeling proteins.

Several additional red fluorescent proteins showing a considerable amount of promise have been isolated from the reef coral organisms. One of the first to be adapted for mammalian applications is HcRed1, which was isolated from Heteractis crispa and is now commercially available. HcRed1 was originally derived from a non-fluorescent chromoprotein that absorbs red light through mutagenesis to produce a weakly fluorescent obligate dimer having an absorption maximum at 588 nanometers and an emission maximum of 618 nanometers. Although the fluorescence emission spectrum of this protein is adequate for separation from DsRed, it tends to co-aggregate with DsRed and is far less bright. An interesting HcRed construct containing two molecules in tandem has been produced to overcome dimerization that, in principle, occurs preferentially within the tandem pairing to produce a monomeric tag. However, because the overall brightness of this twin protein has not yet been improved, it is not a good choice for routine applications in live-cell microscopy.

Developing Monomeric Fluorescent Protein Variants

In their natural states, most fluorescent proteins exist as dimers, tetramers, or higher order oligomers. Likewise, Aequorea victoria green fluorescent protein is thought to participate in a tetrameric complex with aequorin, but this phenomenon has only been observed at very high protein concentrations and the tendency of jellyfish fluorescent proteins to dimerize is generally very weak (having a dissociation constant greater than 100 micromolar). Dimerization of fluorescent proteins has thus not generally been observed when they are expressed in mammalian systems. However, when fluorescent proteins are targeted to specific cellular compartments, such as the plasma membrane, the localized protein concentration can theoretically become high enough to permit dimerization. This is a particular concern when conducting FRET experiments, which can yield complex data sets that are easily compromised by dimerization artifacts.

The construction of monomeric DsRed variants has proven to be a difficult task. More than 30 amino acid alterations to the structure were required for the creation of the first-generation monomeric DsRed protein (termed RFP1). However, this derivative exhibits significantly reduced fluorescence emission compared to the native protein and photobleaches very quickly, rendering it much less useful then monomeric green and yellow fluorescent proteins. Mutagenesis research efforts, including novel techniques such as somatic hypermutation, are continuing in the search for yellow, orange, red, and deep red fluorescent protein variants that further reduce the tendency of these potentially efficacious biological probes to self-associate while simultaneously pushing emission maxima towards longer wavelengths.

Improved monomeric fluorescent proteins are being developed that have increased extinction coefficients, quantum yields, and photostability, although no single variant has yet been optimized by all criteria. In addition, the expression problems with obligate tetrameric red fluorescent proteins are being overcome by the efforts to generate monomeric variants, which have yielded derivatives that are more compatible with biological function.

Perhaps the most spectacular development on this front has been the introduction of a new harvest of fluorescent proteins derived from monomeric red fluorescent protein through directed mutagenesis targeting the Q66 and Y67 residues. Named for fruits that reflect colors similar to the fluorescence emission spectral profile (see Table 1 and Figure 5), this cadre of monomeric fluorescent proteins exhibits maxima at wavelengths ranging from 560 to 610 nanometers. Further extension of this class through iterative somatic hypermutation yielded fluorescent proteins with emission wavelengths up to 650 nanometers. These new proteins essentially fill the gap between the most red-shifted jellyfish fluorescent proteins (such as Venus), and the coral reef red fluorescent proteins. Although several of these new fluorescent proteins lack the brightness and stability necessary for many imaging experiments, their existence is encouraging as it suggests the eventuality of bright, stable, monomeric fluorescent proteins across the entire visible spectrum.

Optical Highlighters

One of the most interesting developments in fluorescent protein research has been the application of these probes as molecular or optical highlighters (see Table 2), which change color or emission intensity as the result of external photon stimulation or the passage of time. As an example, a single point mutation to the native jellyfish peptide creates a photoactivatable version of green fluorescent protein (known as PA-GFP) that enables photoconversion of the excitation peak from ultraviolet to blue by illumination with light in the 400-nanometer range. Unconverted PA-GFP has an excitation peak similar in profile to that of the wild type protein (approximately 395 to 400 nanometers). After photoconversion, the excitation peak at 488 nanometers increases approximately 100-fold. This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell. Illustrated in Figure 6(a) is a transfected living mammalian cell containing PA-GFP in the cytoplasm being imaged with 488-nanometer argon-ion laser excitation before (Figure 6(a)) and after (Figure 6(d)) photoconversion with a 405-nanometer blue diode laser.
Properties of Optical Highlighters
Protein
(Acronym) Excitation
Maximum
(nm) Emission
Maximum
(nm) Molar
Extinction
Coefficient
Quantum
Yield in vivo
Structure Relative
Brightness
(% of EGFP)
PA-GFP 504 517 17,400 0.79 Monomer 41
CoralHue Kaede (G) 508 518 98,800 0.88 Tetramer 259
CoralHue Kaede (R) 572 580 60,400 0.33 Tetramer 59
CoralHue Dronpa (G) 503 518 95,000 0.85 Monomer 240
Kindling (KFP1) 580 600 59,000 0.07 Tetramer 12
PS-CFP (C) 402 468 34,000 0.16 Monomer 16
PS-CFP (G) 490 511 27,000 0.19 Monomer 15
mEosFP (G) 505 516 67,200 0.64 Monomer 128
mEosFP (O) 569 581 37,000 0.62 Monomer 68

Table 2

Other fluorescent proteins can also be employed as optical highlighters. Three-photon excitation (at less than 760 nanometers) of DsRed fluorescent protein is capable of converting the normally red fluorescence to green. This effect is likely due to selective photobleaching of the red chromophores in DsRed, resulting in observable fluorescence from the green state. The Timer variant of DsRed gradually turns from bright green (500-nanometer emission) to bright red (580-nanometer emission) over the course of several hours. The relative ratio of green to red fluorescence can then be used to gather temporal data for gene expression investigations.

A photoswitchable optical highlighter, termed PS-CFP, derived by mutagenesis of a green fluorescent protein variant, has been observed to transition from cyan to green fluorescence upon illumination at 405 nanometers (note photoconversion of the central cell in Figures 6(b) and 6(e)). Expressed as a monomer, this probe is potentially useful in photobleaching, photoconversion and photoactivation investigations. However, the fluorescence from PS-CFP is approximately 2.5-fold dimmer than PA-GFP and is inferior to other highlighters in terms of photoconversion efficiency (the 40-nanometer shift in fluorescence emission upon photoconversion is less than observed with similar probes). Additional mutagenesis of this or related fluorescent proteins has the potential to yield more useful variants in this wavelength region.

Optical highlighters have also been developed in fluorescent proteins cloned from coral and anemone species. Kaede, a fluorescent protein isolated from stony coral, photoconverts from green to red in the presence of ultraviolet light. Unlike PA-GFP, the conversion of fluorescence in Kaede occurs by absorption of light that is spectrally distinct from its illumination. Unfortunately, this protein is an obligate tetramer, making it less suitable fur use as an epitope tag than PA-GFP. Another tetrameric stony coral (Lobophyllia hemprichii) fluorescent protein variant, termed EosFP (see Table 2), emits bright green fluorescence that changes to orange-red when illuminated with ultraviolet light at approximately 390 nanometers. In this case, the spectral shift is produced by a photo-induced modification involving a break in the peptide backbone adjacent to the chromophore. Further mutagenesis of the "wild type" EosFP protein yielded monomeric derivatives, which may be useful in constructing fusion proteins.

A third non-Aequorea optical highlighter, the Kindling fluorescent protein (KFP1) has been developed from a non-fluorescent chromoprotein isolated in Anemonia sulcata, and is now commercially available (Evrogen). Kindling fluorescent protein does not exhibit emission until illuminated with green light. Low-intensity light results in a transient red fluorescence that decays over a few minutes (see the mitochondria in Figure 6(c)). Illumination with blue light quenches the kindled fluorescence immediately, allowing tight control over fluorescent labeling. In contrast, high-intensity illumination results in irreversible kindling and allows for stable highlighting similar to PA-GFP (Figure 6(f)). The ability to precisely control fluorescence is particular useful when tracking particle movement in a crowded environment. For example, this approach has been successfully used to track the fate of neural plate cells in developing Xenopus embryos and the movement of individual mitochondria in PC12 cells.

As the development of optical highlighters continues, fluorescent proteins useful for optical marking should evolve towards brighter, monomeric variants that can be easily photoconverted and display a wide spectrum of emission colors. Coupled with these advances, microscopes equipped to smoothly orchestrate between illumination modes for fluorescence observation and regional marking will become commonplace in cell biology laboratories. Ultimately, these innovations have the potential to make significant achievements in the spatial and temporal dynamics of signal transduction systems.

Fluorescent Protein Vectors and Gene Transfer

Fluorescent proteins are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These ubiquitous probes have been extremely useful as reporters for gene expression studies in cultured cells and tissues, as well as living animals. In live cells, fluorescent proteins are most commonly employed to track the localization and dynamics of proteins, organelles, and other cellular compartments. A variety of techniques have been developed to construct fluorescent protein fusion products and enhance their expression in mammalian and other systems. The primary vehicles for introducing fluorescent protein chimeric gene sequences into cells are genetically engineered bacterial plasmids and viral vectors.

Fluorescent protein gene fusion products can be introduced into mammalian and other cells using the appropriate vector (usually a plasmid or virus) either transiently or stably. In transient, or temporary, gene transfer experiments (often referred to as transient transfection), plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes, but can be expressed in the cytoplasm for a short period of time. Expression of gene fusion products, easily monitored by the observation of fluorescence emission using a filter set compatible with the fluorescent protein, usually takes place over a period of several hours after transfection and continues for 72 to 96 hours after introduction of plasmid DNA into mammalian cells. In many cases, the plasmid DNA can be incorporated into the genome in a permanent state to form stably transformed cell lines. The choice of transient or stable transfection depends upon the target objectives of the investigation.

The basic plasmid vector configuration useful in fluorescent protein gene transfer experiments has several requisite components. The plasmid must contain prokaryotic nucleotide sequences coding for a bacterial replication origin for DNA and an antibiotic resistance gene. These elements, often termed shuttle sequences, allow propagation and selection of the plasmid within a bacterial host to generate sufficient quantities of the vector for mammalian transfections. In addition, the plasmid must contain one or more eukaryotic genetic elements that control the initiation of messenger RNA transcription, a mammalian polyadenylation signal, an intron (optional), and a gene for co-selection in mammalian cells. Transcription elements are necessary for the mammalian host to express the gene fusion product of interest, and the selection gene is usually an antibiotic that bestows resistance to cells containing the plasmid. These general features vary according to plasmid design, and many vectors have a wide spectrum of additional components suited for particular applications.

Illustrated in Figure 7 is the restriction enzyme and genetic map of a commercially available (BD Biosciences Clontech) bacterial plasmid derivative containing the coding sequence for enhanced yellow fluorescent protein fused to the endoplasmic reticulum targeting sequence of calreticulin (a resident protein). Expression of this gene product in susceptible mammalian cells yields a chimeric peptide containing EYFP localized to the endoplasmic reticulum membrane network, designed specifically for fluorescent labeling of this organelle. The host vector is a derivative of the pUC high copy number (approximately 500) plasmid containing the bacterial replication origin, which makes it suitable for reproduction in specialized E. coli strains. The kanamycin antibiotic gene is readily expressed in bacteria and confers resistance to serve as a selectable marker.

Additional features of the EYFP vector presented above are a human cytomegalovirus (CMV) promoter to drive gene expression in transfected human and other mammalian cell lines, and an f1 bacteriophage replication origin for single-stranded DNA production. The vector backbone also contains a simian virus 40 (SV40) replication origin, which is active in mammalian cells that express the SV40 T-antigen. Selection of stable transfectants with the antibiotic G418 is enabled with a neomycin resistance cassette consisting of the SV40 early promoter, the neomycin resistance gene (aminoglycoside 3’-phosphotransferase), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) for messenger stability. Six unique restriction enzyme sites (see Figure 7) are present on the plasmid backbone, which increases the versatility of this plasmid.

Propagation, Isolation, and Transfection of Fluorescent Protein Plasmids

Successful mammalian transfection experiments rely on the use of high quality plasmid or viral DNA vectors that are relatively free of bacterial endotoxins. In the native state, circular plasmid DNA molecules exhibit a tertiary supercoiled conformation that twists the double helix around itself several times. For many years, the method of choice for supercoiled plasmid and virus DNA purification was cesium chloride density gradient centrifugation in the presence of an intercalation agent (such as ethidium bromide or propidium iodide). This technique, which is expensive in terms of both equipment and materials, segregates the supercoiled (plasmid) DNA from linear chromosomal and nicked circular DNA according to buoyant density, enabling the collection of high purity plasmid DNA. Recently, simplified ion-exchange column chromatography methods (commonly termed a mini-prep) have largely supplanted the cumbersome and time-consuming centrifugation protocol to yield large quantities of endotoxin-free plasmid DNA in a relatively short period of time.

Specialized bacterial mutants, termed competent cells, have been developed for convenient and relatively cheap amplification of plasmid vectors. The bacteria contain a palette of mutations that render them particularly susceptible to plasmid replication, and have been chemically permeabilized for transfer of the DNA across the membrane and cell wall in a procedure known as transformation. After transformation, the bacteria are grown to logarithmic phase in the presence of the selection antibiotic dictated by the plasmid. The bacterial culture is concentrated by centrifugation and disrupted by lysis with an alkaline detergent solution containing enzymes to degrade contaminating RNA. The lysate is then filtered and placed on the ion-exchange column. Unwanted materials, including RNA, DNA, and proteins, are thoroughly washed from the column before the plasmid DNA is eluted using a high salt buffer. Alcohol (isopropanol) precipitation concentrates the eluted plasmid DNA, which is collected by centrifugation, washed, and redissolved in buffer. The purified plasmid DNA is ready for duty in transfection experiments.

Mammalian cells used for transfection must be in excellent physiological condition and growing in logarithmic phase during the procedure. A wide spectrum of transfection reagents has been commercially developed to optimize uptake of plasmid DNA by cultured cells. These techniques range from simple calcium phosphate precipitation to sequestering the plasmid DNA in lipid vesicles that fuse to the cell membrane and deliver the contents to the cytoplasm (as illustrated in Figure 8). Collectively termed lipofection, the lipid-based technology has met with widespread acceptance due to its effectiveness in a large number of popular cell lines, and it is now the method of choice for most transfection experiments.

Although transient transfections usually result in the loss of plasmid gene product over a relatively short period of time (several days), stably transfected cell lines continue to produce the guest proteins on a continuous long-term basis (ranging from months to years). Stable cell lines can be selected using antibiotic markers present in the plasmid backbone (see Figure 7). One of the most popular antibiotics for selection of stable transfectants in mammalian cell lines is the protein synthesis-inhibiting drug G418, but the required dose varies widely according to each cell line. Other common antibiotics, including hydromycin-B and puromycin, have also been developed for stable cell selection, as have genetic markers. The most efficient method of obtaining stable cell lines is to employ a high efficiency technique for the initial transfection. In this regard, electroporation has proven to generate stable transfectants with linearized plasmids and purified genes. Electroporation applies short, high voltage pulses to a cellular suspension to induce pore formation in the plasma membrane, subsequently allowing the transfection DNA to enter the cell. Specialized equipment is necessary for electroporation, however, the technique is comparable in expense to lipofection reagents when a large number of transfections are performed.

The Future of Fluorescent Proteins

The focus of current fluorescent protein development is centered on two basic goals. The first is to perfect and fine-tune the current palette of blue to yellow fluorescent proteins derived from Aequorea victoria jellyfish, while the second aim is to develop monomeric fluorescent proteins emitting in the orange to far red regions of the visible light spectrum. Progress toward these goals has been quite impressive, and it is not inconceivable that near-infrared fluorescent proteins loom on the horizon.

The latest generation of jellyfish variants has solved most of the deficiencies of the first generation fluorescent proteins, particularly for the yellow and green derivatives. The search for a monomeric, bright, and fast-maturing red fluorescent protein has resulted in several new and interesting classes of fluorescent proteins, particularly those derived from coral species. Development of existing fluorescent proteins, together with new technologies, such as insertion of unnatural amino acids, will further expand the color palette. As optical spectral separation techniques become better developed and more widespread, these new varieties will supplement the existing palette, especially in the yellow and red regions of the spectrum.

The current trend in fluorescent probe technology is to expand the role of dyes that fluoresce into the far red and near infrared. In mammalian cells, both autofluorescence and the absorption of light are greatly reduced at the red end of the spectrum. Thus, the development of far red fluorescent probes would be extremely useful for the examination of thick specimens and entire animals. Given the success of fluorescent proteins as reporters in transgenic systems, the use of far red fluorescent proteins in whole organisms will become increasingly important in the coming years.

Finally, the tremendous potential in fluorescent protein applications for the engineering of biosensors is just now being realized. The number of biosensor constructs is rapidly growing. By using structural information, development of these probes has led to improved sensitivity and will continue to do so. The success of these endeavors certainly suggests that almost any biological parameter will be measurable using the appropriate fluorescent protein-based biosensor.

BACK TO FLUORESCENCE MICROSCOPY

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232.

George H. Patterson and Jennifer Lippincott-Schwartz - Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.
Nathan S. Claxton and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

GFP, RFP

2005-10-11T20:12:00.000-07:00

This web page was produced as an assignment for an undergraduate course at Davidson College
Red Fluorescent Protein

Figure 1. Image of DsRed (red fluorescent protein) and
and green fluorescent protein (GFP). Image used with
permission from Clontech.

Reporter genes are valuable tools in molecular biology because they allow researchers to visualize the protein products of genes. Green fluorescent protein (GFP) is unique because it naturally fluoresces with out the addition of substrates or enzymes, allowing researchers to view genetic expression in living cells. GFP can serve as a transcription marker or it can be used to visualize protein localization. The newly discovered red fluorescent protein (RFP) shares many properties of GFP and can be applied in a similar manner. One form of red fluorescent protein (DsRed), isolated from Discosoma striata and manufactured by Clontech, has an optimal absorption at 558 nm and emits light at 583 nm (CLONTECH, 1999). Another form of RFP (cob A) has been isolated from Propionibacterium freudenreichii (Wildt and Deuschle, 1999). One advantage of red fluorescent protein (RFP) is that it produces less background interference than GFP. Although the greatest advantage of RFP is that it can be used in conjunction with GFP to colabel cells.

Expression System

Cloning and Fusion
RFP is a reporter gene that allows the visual detection of gene expression in living cells. This is how it works. First the RFP gene must be fused to a gene of interest. A commercially bought RFP gene is already inserted into a plasmid (there are various types of plasmids available that have been optimized for different types of research). RFP gene plasmids (pRFP) have polylinkers located near the RFP gene. The gene of interest and pRFP are cut with a restriction enzyme and mixed together so they can ligate. Once the gene of interest is ligated to the RFP plasmid, it can be cloned by inserting the pRFP into bacterial cells. Depending on the plasmid being used, an ampR gene or another gene conferring resistance may also be found on pRFP. Thus transformed bacteria can be selected by their resistance to a substance. Again depending on the type of pRFP purchased, a promoter many need to be inserted into the plasmid or the plasmid may already contain a promoter. The plasmid can now be cloned via bacterial replication.

Expression
The vectors created in the previous steps can be inserted into any living cells. Once inside, the chimeric protein will be expressed through the natural process of transcription and translation. This protein should contain a functional RFP and protein of interest. A light of the appropriate wavelength must be shown on the cells and the RFP will fluoresce, allowing visual detection of the protein of interest.

Plasmids available from Clontech

Figure 2. Plasmids containing the RFP gene that are currently available from Clontech.
Image used with permission from Clontech.

pDsRed: This is a cDNA copy of the RFP gene (DsRed). It is flanked by two polylinkers (MCS-multiple cloning sites). This plasmid also contains ampR and the lac promoter. Thus the RFP gene can be removed and inserted into a new plasmid or this plasmid can be inserted into the appropriate vector. This plasmid is optimal for nonmammalian expression.

pDsRed1-1: This plasmid contains an RFP gene that has been altered for human expression of RFP (DsRed1). A promoter must be inserted into this plasmid for RFP to be expressed. Rather than create a fusion protein, this vector is ideal for research on promoter and enhancer sequences in mammals.

pDsRed1-N1: This plasmid also contains DsRed1 as well as a CMV promoter, making it ideal for creating fusion proteins to be expressed in mammalian cells.

Microscopy
The expression of RFP can be visualized using a fluorescence microscope with a rhodamine or FITC filter set (Wildt and Deuschle, 1999). Colabelling is possible by transfecting cells with different plasmids each containing one fluorescent protein gene and one gene of interest (Figure 3). Double or even triple labeling a cell is a highly informative tool. Now researchers can see the interaction between gene products or the difference in localization between two or more protiens. Another possible experiment is to monitor the progressional expression of two or more genes, which would be very useful in developmental studies.

Figure 3. HeLa Cell transfected with three fusion fluorescent
proteins. Nuclear proteins fused with enhanced cyan fluorescent
protein (ECFP). Tubulin proteins labeled with EY(yellow) FP.
Mitochondrial proteins were labeled with DsRed. Image used with
permission from Clontech.

Flow Cytometry
Flow cytometry allows a mixed sample of cells to be sorted their physical or chemical properties. Cells that are unusual in some way can be collected for further research. Cells are labeled with a fluorescent dye that can be excited by a beam of light. Cells that emit fluorescence are detected and this information is stored for further study. In some flow cytometry devices fluorescing cells are sequestered. DsRed serves as such a dye. A 488nm argon laser can be used to excite DsRed in flow cytometry experiments (CLONTECH, 1999).

All questions concerning the purchase of this product should be directed to Clontech
Works Consulted

Campbell, N. A. 1996. Biology, 4th ed. Menlo Park, CA: Benjamin/Cummings Publishing Company, Inc., . p. 372-374.

CLONTECH Laboratories. 1999. Living Colors Red Fluorescent Protein. CLONTECHniques.October:2-6.
accessed 2000 13 Feb.

The John Curtin School of Medical Research. 1998 March 12. JCSMR Flow Cytometry "An introduction".
.Accessed 2000 20 Feb.

Wildt, S and Deuschle, U. 1999. cobA, a red fluorescent transcriptional reporter for Escherichia
coli, yeast, and mammalian cells. Nature Biotechnology. 17: 1175-1178.
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© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: jereynoldskenneally@davidson.edu

Dairy Science and Technology Home Page

2005-09-26T21:59:00.000-07:00

Is glass liquid or solid?

2005-09-16T01:17:00.000-07:00

Is glass liquid or solid?

Is glass liquid or solid?

It is sometimes said that glass in very old churches is thicker at the bottom than at the top because glass is a liquid, and so over several centuries it has flowed towards the bottom. This is not true. In Mediaeval times panes of glass were often made by the Crown glass process. A lump of molten glass was rolled, blown, expanded, flattened and finally spun into a disc before being cut into panes. The sheets were thicker towards the edge of the disc and were usually installed with the heavier side at the bottom. Other techniques of forming glass panes have been used but it is only the relatively recent float glass processes which have produced good quality flat sheets of glass.

To answer the question "Is glass liquid or solid?" we have to understand its thermodynamic and material properties.

Thermodynamics of glass

There is still much about the molecular physics and thermodynamics of glass that is not well understood, but we can give a general account of what is thought to be the case.

Many solids have a crystalline structure on microscopic scales. The molecules are arranged in a regular lattice. As the solid is heated the molecules vibrate about their position in the lattice until, at the melting point, the crystal breaks down and the molecules start to flow. There is a sharp distinction between the solid and the liquid state, that is separated by a first order phase transition, i.e. a discontinuous change in the properties of the material such as density. Freezing is marked by a release of heat known as the heat of fusion.

molecular arrangement in a crystal

A liquid has viscosity, a measure of its resistance to flow. The viscosity of water at room temperature is about 0.01 poises. A thick oil might have a viscosity of about 1.0 poise. As a liquid is cooled its viscosity normally increases, but viscosity also has a tendency to prevent crystallisation. Usually when a liquid is cooled to below its melting point, crystals form and it solidifies; but sometimes it can become supercooled and remain liquid below its melting point because there are no nucleation sites to initiate the crystallisation. If the viscosity rises enough as it is cooled further, it may never crystallise. The viscosity rises rapidly and continuously, forming a thick syrup and eventually an amorphous solid. The molecules then have a disordered arrangement, but sufficient cohesion to maintain some rigidity. In this state it is often called an amorphous solid or glass.

molecular arrangement in a glass

Some people claim that glass is actually a supercooled liquid because there is no first order phase transition as it cools. In fact, there is a second order transition between the supercooled liquid state and the glass state, so a distinction can still be drawn. The transition is not as dramatic as the phase change that takes you from liquid to crystalline solids. There is no discontinuous change of density and no latent heat of fusion. The transition can be detected as a marked change in the thermal expansivity and heat capacity of the material.

The temperature at which the glass transition takes place can vary according to how slowly the material cools. If it cools slowly it has longer to relax, the transition occurs at a lower temperature and the glass formed is more dense. If it cools very slowly it will crystallise, so there is a minimum limit to the glass transition temperature.

Density as a function of temperature
in the phases of glassy materials

A liquid to crystal transition is a thermodynamic one; i.e. the crystal is energetically more favourable than the liquid when below the melting point. The glass transition is purely kinetic: i.e. the disordered glassy state does not have enough kinetic energy to overcome the potential energy barriers required for movement of the molecules past one another. The molecules of the glass take on a fixed but disordered arrangement. Glasses and supercooled liquids are both metastable phases rather than true thermodynamic phases like crystalline solids. In principle, a glass could undergo a spontaneous transition to a crystalline solid at any time. Sometimes old glass devitrifies in this way if it has impurities.

The situation at the level of molecular physics can be summarised by saying that there are three main types of molecular arrangement:

crystalline solids: molecules are ordered in a regular lattice
fluids: molecules are disordered and are not rigidly bound.
glasses: molecules are disordered but are rigidly bound.

[Just to illustrate that no such classification could ever be complete, recently scientists have succeeded in making quasi-crystals that are quasi-periodic. They do not fit into the above scheme and are sometimes described as being halfway between crystals and glass.]

It would be convenient if we could conclude that glassy materials changed from being a supercooled liquid to an amorphous solid at the glass transition, but this is very difficult to justify. Polymerised materials such as rubber show a clear glass transition at low temperatures but are normally considered to be solid in both the glass and rubber conditions.

It is sometimes said that glass is therefore neither a liquid nor a solid. It has a distinctly different structure with properties of both liquids and solids. Not everyone agrees with this terminology.

Material properties of glasses

Usually when people talk about solids and liquids, they are referring to macroscopic material properties rather than the arrangement of molecules. After all, glass as a material was known about long before its molecular physics was understood. Macroscopically, materials exhibit a very wide range of behaviours. Solids, liquids and gases are ideal behaviours characterised by properties such as compressibility, viscosity, elasticity, strength and hardness. But materials don't always behave according to such ideals. For example, it's possible to take water from being a liquid to a gas at high pressure without its passing through a phase transition; so at some stage it must be between an ideal liquid and an ideal gas.

For crystalline substances the distinction between the solid and liquid states is very clear, but what about glasses? Indeed, where do polymers, gels, foams, liquid crystals, powders and colloids fit into this picture? Some people say that there is no clear distinction between a solid and a liquid in general. A solid, they claim, should just be defined as a liquid with a very high viscosity. They set an arbitrary limit of 1013 poises above which they say it's a solid and below which it's a liquid.

According to another point of view, this ignores a distinction between viscosity of liquids and plasticity of solids. An ideal Newtonian liquid deforms at a rate which is proportional to stresses applied and its viscosity. For arbitrarily small stresses a viscous liquid will flow. Molasses, pine pitch and Silly Putty are examples of liquids with very high viscosity which flow very slowly under only the force of their own weight. On the other hand, plastics can be very soft but are still considered solid because they have rigidity and do not flow.

Solids are elastic when small stresses are applied. They deform but return to their original shape when the stress is removed. When higher stresses are applied some solids break while others exhibit plasticity. Plasticity means that they deform and don't return to their original shape when the stress is removed. Many substances including metals such as copper have plasticity. The resistance to flow under plastic deformation is called its viscoplasticity. This is like viscosity, except that there's a minimum stress known as the elastic limit below which there is no plasticity. Materials with plasticity do not flow, but they may creep, meaning they deform slowly but only when held under constant stress.

So an arbitrary measure of viscosity or viscoplasticity is not a good way to distinguish solids from liquids. Another way to define the distinction between solid and liquid is to say that, if there is a minimum shear stress required to produce a permanent deformation then it is a solid. This is just a precise way of saying it has some rigidity. A liquid can then be defined as a material that will flow. If it is placed in a container it will eventually flow to fill the lower reaches until its own surface is flat. The difficulty is that these two definitions do not cover all cases. There are materials that have some limited flow known as viscoelasticity. The material will deform elastically under stress. If the stress is held for a long time, the deformation becomes permanent even if the stress was small. Materials with viscoelasticity may seem to flow slowly for a while but then stop. It is futile to try to make a clear cut distinction between liquids and solids in cases of such behaviour.

Types of Glass

To be sure that glass in old windows has not flowed, we need to recognise the different properties of different glasses. Glass can be made from pure silica, but fused silica has a high glass transition point at around 1200° C which makes it difficult to mould into panes or bottles. At least 2000 years ago it was learned how to lower the softening temperature by adding lime and soda before heating, which resulted in a glass containing sodium and calcium oxides. Soda-lime glass used for windows and bottles today contains other oxides as well. Measuring the glass transition temperature for different glasses is not easy because it changes according to how slowly the glass is cooled. In the case of modern soda-lime glass, a quick cooling will produce a glass transition at about 550° C. There is thought to be a minimum glass transition temperature at about 270° C, and if it is cooled very slowly it can still be a supercooled liquid down to just above that temperature. Glass such as Pyrex (used for test-tubes and ovenware) is usually based on boro-silicates or alumino-silicates, which withstand heating better and typically have a higher glass transition temperature. Some glasses, such as the leaded variety, have lower transition temperatures.

Sometimes people say that good evidence that glass does not flow is provided by telescope lenses which after 150 years still maintain excellent optical qualities. They would be spoiled by the slightest deformation. In fact, optical glass is usually not the same as the glass used in windows and bottles. It may be based on boro-silicate or soda-lime glass with other metallic oxides added to improve its thermal and optical properties. So old telescope lenses and mirrors provide good evidence that some glasses do not flow, but little evidence to support the claim that glass in old windows has not flowed. Another example is stone age arrow heads made of obsidian, a natural glass. These are found to be still razor sharp after tens of thousands of years, but again, this glass is mainly silica and alumino-silicates and is much tougher than window glass.

For definitive evidence that glass has not flowed in old windows we must examine the oldest examples. Early glass used to make bottles and windows was usually formed by adding soda and lime to silicates. Sometimes potash was added instead. Usually there were other impurities which made it softer than modern soda-lime glass. Other compounds were often added to give colour or to improve its properties. The Romans were making glass objects of this sort in the 1st century AD, and despite being very delicate, some examples remain--such as the elaborately decorated Portland Vase kept at the British Museum. Roman glassware provides some of the best available evidence that types of soda-lime glass are not fluid, even after nearly 2000 years. The oldest remaining examples of stained glass windows that remain in place have lasted since the 12th century. The oldest of all are the five figures in the clerestory of Augsburg Cathedral in Germany, which are dated to between 1050 to 1150. Many other early examples are found in France and England including the magnificent North Rose window of Notre Dame, Paris dating from 1250.

There have been many claims (especially by tour guides) that such glass is deformed because the glass has flowed slowly over the centuries. This has become a persistent myth, but close inspection shows that characteristic signs of flow, such as flowing around, and out of the frame, are not present. The deformations are more consistent with imperfections of the methods used to make panes of glass at the time. In some cases gaps appear between glass panes and their frames, but this is due to deformations in the lead framework rather than the glass. Other examples of rippling in windows of old homes can be accounted for because the glass was imperfectly flattened by rolling before the float glass process came into use.

It is difficult to verify with absolute certainty that no examples of glass flow exist, because there are almost always no records of the original state. In rare cases stained glass windows are found to contain lead which would lower the viscosity and make them heavier. Could these examples deform under their own weight? Only careful study and analysis can answer this question. Robert Brill of the Corning glass museum has been studying antique glass for over 30 years. He has examined many examples of glass from old buildings, measuring their material properties and chemical composition. He has taken a special interest in the glass flow myth and has always looked for evidence for and against. In his opinion, the notion that glass in Mediaeval stained glass windows has flowed over the centuries is untrue and, he says, examples of sagging and ripples in old windows are also most likely physical characteristics resulting from the manufacturing process. Other experts who have made similar studies agree. Theoretical analysis based on measured glass viscosities shows that glass should not deform significantly even over many centuries, and a clear link is found between types of deformation in the glass and the way it was produced.

Conclusion

There is no clear answer to the question "Is glass solid or liquid?". In terms of molecular dynamics and thermodynamics it is possible to justify various different views that it is a highly viscous liquid, an amorphous solid, or simply that glass is another state of matter which is neither liquid nor solid. The difference is semantic. In terms of its material properties we can do little better. There is no clear definition of the distinction between solids and highly viscous liquids. All such phases or states of matter are idealisations of real material properties. Nevertheless, from a more common sense point of view, glass should be considered a solid since it is rigid according to every day experience. The use of the term "supercooled liquid" to describe glass still persists, but is considered by many to be an unfortunate misnomer that should be avoided. In any case, claims that glass panes in old windows have deformed due to glass flow have never been substantiated. Examples of Roman glassware and calculations based on measurements of glass visco-properties indicate that these claims cannot be true. The observed features are more easily explained as a result of the imperfect methods used to make glass window panes before the float glass process was invented.

Farms For City Children Edu tours

2005-08-23T01:16:00.000-07:00

Farms For City Children

Farms For City Children
» motherhood
This list was posted at the Asia Parent List some time back. I know the school holidays are over, but there are always weekends to bring the kids to visit some farms and see some animals, and also buy some fresh farm produce to cook dinner :)

1) Hay Dairies

goat.JPGTel: 67920931
Add: 3 Lim Chu Kang Lane 4
Open: 9-4pm daily
Milking time: Between 9-10am

Purchase healthful fresh goat's milk – it comes in plain or chocolate flavours – or pet the 200-odd goats and kids. Milking demo are on between 9am and 10am at half-hour intervals. Tours can be arranged at $3/person, including a free bottle of mild and a souvenir.

2) Dairy Technology Singapore Pte Ltd

Tel: 67937769
Add: 8 Lim Chu Kang Lane 8A
Open: 10-5pm daily

See the daily operations of a dairy farm and feed the adorable calves. The tour includes a visit to a milking parlour. While commercial milking is done mechanically, you can ask to try hand milking. The 45-mins tour costs $3/person and includes a free bottle of milk. Call to book the tour 2 weeks in advance.

3) Nippon Koi Farm

koi.jpgTel: 67631882
Add: 45 Jalan Lekar
Open: 9-7pm daily

A must for koi-lovers. These ornamental fishes are regarded as "fengshui" fishes' supposedly able to bring good fortune to their owners. Nippon Koi Farm is where u can see and purchase award- winning Japanese carp or koi. Free entry for all and kids get to feed the koi for free too. The farm also runs a koi hotel and has a Luohan pavilion for viewing.

4) Farmart Centre

vegefarm_home.jpgTel: 67670070 / 67640328
Add: 67 Sungei Tengah Rd
Open: Food Outlets (10-10pm fm Mon to Sat), (9-10pm on Sun & PH)
Retail Outlets (10-8pm fm Mon to Sat), (9-8pm on Sun & PH)
Guided Tour Price:
External Farms $12/person. I
Indoor Farms $4/person

Farms, and all under one roof! A one-stop farm estate selling and displaying almost everything agriculture fm vege, herbs & spices, birds' nest, seafood, ornamental fish, poultry and eggs, lotuses and orchids and even puppies, as well as Singapore's only bee farm. Leave the kids to enjoy doing arts and crafts at a play corner with a playground or the animal corner where they can feed the Himalayan goats and fish for guppies. Catch yourselves some prawns at the prawn pond and if u like, enjoy them straightaway at the barbecue pits on site. Purchase some croc or ostrich meat if u have the stomach for exotic food, or regular fare at the café or seafood restaurant. Hop onto a free shuttle service from Choa Chu Kang Interchange (redeem the shuttle fee of 50 cents against purchases at the centre).

5) Qian Hu Fish Farm

qianhu3.jpgTel: 67667087
Add: 71 Jalan Lekar
Open: 9-6pm (Mon-Thu), 9-7pm (Fri-Sun and PH)
Guided Tour w/Souvenir
Adult: $2.50 Child: $1.50
Guided Tour w/ Souvenir & Longkang Fishing
Adult: $7.50 Child: $5.50

The farm's specialty is the dragon-fish (arowana). Go and see for yourself a fully-grown one can fetch a whopping $50,000. Kids will spend ages in a special feeding corner. For $5, you can try longkang fishing, feed the koi, rabbits, guinea pigs, chickens, ducks and geese. A wishing pond is home to a family of tortoises. Guided tours for a min. of 30 pax must be arranged a week in advance. Free shuttle svc runs to and fm Choa Chu Kang MRT.

6) Jurong Crocodile & Reptile Paradise

Tel: 62618866
Add: 241 Jalan Ahmad Ibrahim
Open: 9-6pm daily
Fees: Adult ($7) Child-below 12 yrs ($3.50)

Jus opp the Jurong Bird Park. Get up close and personal with 3000
of different species of reptiles in shows, photo-taking and feeding
sessions. With regular crocodile shows throughout the day and an
underwater observation gallery, you can observe these creatures.
The Cavern of Darkness allows u to watch the crocs in a simulated
Asian tropical jungle complete with life-like sounds in the creepy
shadows of the night.

7) Jurong Frog Farm

frogfarm3.gifTel: 67917229
Add 56 Lim Chu Kang Lane 6
Open: 7-6pm daily

This is the only frog-breeding farm in S'pore. They rear American ullfrogs believed to have medical value. You can book a guided tour a week in advance, although walk-ins are also welcome. For $3.50, u'll receive a half-hour tour of the farm, even to the culling station if u request for it. Tour includes a free bottle of their own Essence of Bullfrog with ginseng and cordyceps. Visitors can purchase packed frogs at $10 per 500gm.

8) Aero-Green Technology (S) Pte Ltd

Tel: 67924298 / 68619286
Add 115 Neo Tiew Crescent
Open: 9-6pm daily

Buy the freshest veg at cheaper prices. This farm produces abt 500kg of aeroponic veg every day – Bak Choy, Choy Sum and Kai Lan, as well as butterhead lettuce, cherry tomatoes, herbs, fruit and more. The farm sometimes conducts workshops and cooking classes – call to check on schedules. Conducted tours which include comprehensive educational coverage on the plants grown on the farm are avail. For groups of at least 20 and can be arranged at $6/person.. Book at least 2 wks in advance.

9) Seng Choon Egg Farm Pte Ltd

Tel: 67922858
Add 109 Sungei Tengah Road
Open: 8-5pm (Mon-Fri), 8-12pm (Sat)

This modern egg farm has abt 500,000 chickens and can produce up to 250,000 fresh eggs daily. It is completely high tech – everything from feeding the chickens to collecting the eggs is automated and computerized. Free visits need to be booked in advance with a min. of 30 persons per grp. At press time, farm was temporarily close to public.

10) Lian Wah Hang Quail Farm

quail.jpgTel: 67921366
Add 5 Lim Chu Kang Lane 6F
Open: 10-4pm (Mon to Sat), 11-4 (Sun)

See quails, partridges, geese, guinea fowl, pigeons and cassowary. Owner and host William Ho is a friendly guy who will tell u all abt his animals and the quail egg production process. Hard-boiled quail eggs are complimentary at the end of each tour between Mon and Sat. Sun tours include a `happy hour' between 11-3pm. Buy fresh quail eggs at farm prices. At press time, the farm was temporarily shut, but you can stil meet William for his entertaining bird talk every weekend at Farmart Centre new Farmers' Market. See Farmart Centre.

(list compiled by Magdalene)

11) Poison Ivy and Bollywood Vege (further down the road)

Tel : 6898 5001
Add : 100 Neo Tiew Rd Singapore 719026

S$2 / person entry into farm
Arrive at about 11am to "chope" seats and then walk around the farm with
kids as lunch crowd drifts in at noon at Posion Ivy.

Bollywood Vege is nearby. It's an organic vegetable farm with lots of trees and plants.

12)Petals & Leaves

Tel : 6793 8878
Add: 240, Neo Tiew Crescent.

It's under the Nyee Phoe Group, serving Nonya food. Look for Edwin Pereira to make reservations. Make reservations as they do run out of food.

Please call up the relevant farm and check the opening hours if you do plan to visit. Information posted here might not be accurate due to fatigue or typo errors, and no. 11 and 12 are from hearysay

I have only visited the Farmart. Perhaps it was a slow weekday, it was almost empty except for us and the shop owners. I like the concept of a farmart, but it seems unable to attract locals. Maybe we are too comfortable with our super markets, and the variety of farm products we can find there is limited too. Well, it is better to visit farmart during the weekend for more action, especially if you plan to get some farm produce.
Posted by hait on 1:42pm :: add new comment | trackback
Comments 2
There is a really nice farm at Seletar way called the Animal Resort. The children gets to feed various animals like chickens, ducks, rabbits, fish and even a horse! My eldest dotter, Alyssa enjoyed the trip so much that she always remember feeding the horse even though she went there at 2+ yrs old . We should organise a trip there one day, the little ones will be thrilled :)

3D Rendered Pictures Of My Ballista

2005-08-10T21:07:00.000-07:00

3D Rendered Pictures Of My Ballista

- Formulas making your own homemade glue

2005-07-26T19:26:00.000-07:00

- Formulas making your own homemade glue

Detection

2005-07-11T20:53:00.000-07:00

Synders Test Agar Cavity
Prone

Science Explorer: Super Sparker--make very tiny lightning anytime!

2005-07-11T20:29:00.000-07:00

Science Explorer: Super Sparker--make very tiny lightning anytime!

Physics Today November 2002

2005-07-11T20:27:00.000-07:00

Physics Today November 2002

Darwinian gastronomy: Why we use spices

2005-06-24T03:00:00.000-07:00

Darwinian gastronomy: Why we use spices

ProQuest(R) Help
Darwinian gastronomy: Why we use spices
Bioscience; Washington; Jun 1999; Paul W Sherman;Jennifer Billing;

Volume: 49
Issue: 6
Start Page: 453-463
ISSN: 00063568
Subject Terms: Spices
Flowers & plants
Food contamination & poisoning
Abstract:
Humans have borrowed plants' chemical "recipes" for evolutionary survival for use in cuisine to combat foodborne microorganisms and to reduce food poisoning. This explains the use of spices.

Full Text:
Copyright American Institute of Biological Sciences Jun 1999
[Headnote]
Spices taste good because they are good for us

Spices are plant products used in flavoring foods and beverages. For thousands of years, aromatic plant materials have been used in food preparation and preservation, as well as for embalming, in areas where the plants are native, such as Hindustan and the Spice Islands (Govindarajan 1985, Dillon and Board 1994). During and after the Middle Ages, seafarers such as Marco Polo, Ferdinand Magellan, and Christopher Columbus undertook hazardous voyages to establish routes to trading ports in primary spice-growing regions (Parry 1953). The spice trade was so crucial to national economies that rulers repeatedly mounted costly expeditions to raid spice-growing countries, and struggles for the control of these countries precipitated several wars. When Alarich, a leader of the Goths, laid siege to Rome in AD 408, he demanded as ransom various precious metals and 3000 pounds of pepper (Scheiper 1993).

Today, spice use is ubiquitous, but spices are far more important in some cuisines than others. Most people have experienced this variability firsthand, when traveling in foreign lands, dining at international restaurants, or preparing exotic recipes at home. Japanese dishes are often "delicate," Indonesian and Szechwan dishes "hot," and middle European and Scandinavian dishes "bland." Usually these differences are merely chalked up to cultural idiosyncrasies. Several years ago, we became curious about this interpretation. We wondered if there are any predictable patterns of spice use and, if so, what factors might underlie them. In this article, we summarize the results of our inquiries. We found that spice use is decidedly nonrandom and that spices have several beneficial effects, the most important of which may be reducing foodborne illnesses and food poisoning.

What is a spice?

"Spice" is a culinary term, not a botanical category-it does not refer to a specific kind of plant or plant part (Farrell 1990). Indeed, spices come from various woody shrubs and vines, trees, aromatic lichens, and the roots, flowers, seeds, and fruits of herbaceous plants (Figure 1). Cookbooks generally distinguish between seasonings (spices used in food preparation) and condiments (spices added after food is served), but not between herbs and spices. However, herbs, which are defined botanically (as plants that do not develop woody, persistent tissue), usually are called for in their fresh state, whereas spices generally are dried (Figure 2). Salt is sometimes thought of as a spice, but it is a mineral.

Each spice has a unique aroma and flavor, which derive from compounds known as phytochemicals or "secondary compounds" (because they are secondary to the plant's basic metabolism). These chemicals evolved in plants to protect them against herbivorous insects and vertebrates, fungi, pathogens, and parasites (Fraenkel 1959, Walker 1994). Most spices contain dozens of secondary compounds. These are plants' recipes for survival-legacies of their coevolutionary races against biotic enemies.

Patterns of spice use

Conventional wisdom tells us that cuisines of tropical countries are spicier than those of northern countries, but patterns of spice use around the world have not been quantified. To do so, we located "traditional" cookbooks, which were written primarily to archive the author's native cuisine. We analyzed recipes in 93 traditional cookbooks from 36 counties (at least two books from each country) and quantified the use of 43 spices in these countries (Table 1).

In gathering our data, we did not distinguish between seasonings and condiments or between herbs and spices. We focused on meat-based recipes (those in which at least onethird of the volume or weight consisted of meat) rather than vegetablebased recipes for two reasons. First, traditional cookbooks have many more meat-based dishes than vegetarian dishes, enabling us to obtain adequate sample sizes (Table 1). Second, unrefrigerated meats spoil faster than vegetables and are more often associated with foodborne disease outbreaks (Sockett 1995). Thus, any relationship between spoilage and spice use should be more apparent in meatbased than vegetable-based recipes.

In summarizing the data, we encountered two problems. The first was whether or not to treat onions (Allium cepa: chives, leeks, and shallots) and chilis (Capsicum frutescens: capsaicin-containing peppers) as spices. Although these plants are often used solely as spices, they are also served as main dishes. Following the lead of previous authors (e.g., Farrell 1990, Tainter and Grenis 1993, Hirasa and Takemasa 1998), we decided to include both plants as spices because, regardless of the quantities called for, they always contribute their phytochemicals to the cuisine. The second problem was how to treat the comparative information statistically, because not all countries are equally "independent" (e.g., due to shared ancestry or recent immigration). However, because it is unclear how to assess independence of a specific cultural practice, such as spice use, and because our sample was so broad (representing every continent and 16 of the world's 19 major linguistic groups [Ruhlen 1987]), we treated all countries as if they were independent and used nonparametric analyses.

We tabulated the ingredients in 4578 meatbased recipes and discovered that most of them (93%) call for at least one spice. On average, recipes called for 3.9 + 1.7 (SD) spices, although some lacked spices entirely and others had up to 12 spices. In 10 countries-Ethiopia, Kenya, Greece, India, Indonesia, Iran, Malaysia, Morocco, Nigeria, and Thailand-every meatbased recipe we examined called for at least one spice. whereas in Scandinavian countries one-third of the recipes did not call for any spices.
[Table]
Table 1.

The frequency of use of individual spices also varied widely (Figure 3). Black pepper and onion were called for most frequently, in 63 % and 65 % of all meat-based recipes, respectively. Other commonly used spices included garlic (35% of recipes), chilis (24%), lemon and lime juice (23%), parsley (22%), ginger (16%), and bay leaf (13%). However, the majority of spices were used infrequently. Of the 43 spices we analyzed, 35 (81%) were used in less than 10% of the recipes, and 29 (67%) were used in less than 5% of the recipes.

Antimicrobial properties of spices

Why are spices used? The obvious answer is that they enhance food flavor, color, and palatability. Of course this is true as far as it goes. However, such a proximate (immediate cause) explanation does not address the ultimate (evolutionary) questions of why cuisines that contain pungent plant products appeal to people and why some phytochemicals are tastier than others. Answers to proximate and ultimate questions are complementary, not mutually exclusive, and full understanding requires explanations at both "levels of analysis" (Sherman 1988).o analysis" (Sherman A clue to the ultimate reason for spice use may lie in the protective effects of phytochemicals against plants' biotic enemies. After all, meat and other food items are also attacked by bacteria and fungi, indeed by some of the same species that afflict plants. Throughout recorded history, foodborne bacteria (especially species of Clostridium, Escherichia, Listeria, Salmonella, Shigella, and Vibrio) or their toxins have been serious health concerns, and they still are (Hui et al. 1994, WHO 1996). If spices were to kill such microorganisms or inhibit their growth before they could produce toxins, use of spices might reduce foodborne illnesses and food poisoning (Billing and Sherman 1998). If this antimicrobial hypothesis were true, several predictions should be fulfilled.

Prediction 1. Spices should exhibit antibacterial and antifungal activity. Microbiologists and food-product developers have conducted laboratory experiments that involve challenging numerous foodborne bacteria, fungi, and yeasts with phytochemicals extracted from spice plants. Multiple techniques have been used to investigate inhibition, and the primary data vary considerably in quality and quantity for different spices. Nevertheless, it is now clear that many spices have potent antimicrobial properties (e.g., Hargreaves et al. 1975, Shelef 1984, Deans and Ritchie 1987, Zaika 1988, Beuchat 1994, Nakatani 1994, Hirasa and Takemasa 1998).

We were particularly interested in the ability of spices to inhibit bacteria because bacteria are more commonly incriminated in foodborne disease outbreaks than yeasts or fungi (Varnam and Evans 1991, Todd 1994). All 30 spices for which we located laboratory test results were found at some concentration to kill or inhibit at least 25% of the bacterial species on which they had been tested, and 15 of these spices inhibited at least 75% of bacterial species (Figure 4). Garlic, onion, allspice, and oregano were found to be the most potent spices: They inhibited or killed every bacterium they were tested on. Most of the tested microorganisms are widely distributed geographically, so they have the potential to contaminate foods everywhere.

Prediction 2. Use of spices should be greatest in hot climates, where unrefrigerated foods spoil especially quickly. Uncooked meats and meat dishes that are prepared in advance and stored at room temperatures for more than a few hours typically build up massive bacterial populations, especially in tropical climates (Hobbs and Roberts 1993). Therefore, we used each country's average annual temperature as a relative indicator of its rate of meat spoilage. Our test assumes that traditional meat-based recipes were developed before widespread refrigeration. We cannot directly evaluate this assumption because the cookbooks we examined rarely discussed the history of individual dishes. However, the assumption seems reasonable because any recipe that has been around for more than five generations (approximately 100 years) would pre-date electrical refrigeration. Most of the recipes we examined probably were at least that old.

We used climate atlases (e.g., Bair 1992) to tabulate information on mean temperatures in all 36 countries (Table 1). Temperatures ranged from 2.8 oC (Norway) to 27.6 oC (Thailand). Consistent with the prediction, we found that as average temperatures increased among countries, there were significant increases in the fraction of recipes that called for at least one spice, the mean numbers of spices per recipe, and the numbers of different spices used (Figure 5). For example, India's cuisine included 25 different spices, of which an average of 9.3 were called for per recipe, whereas Norwegian cuisine included only 10 different spices and called for an average of 1.6 per recipe. In Hungary, which has a temperate climate, the cuisine included 21 spices, of which an average of 3.0 were called for per recipe.
[Photograph]
Figure 1.
[Photograph]
Figure 2.

The relative use of many individual spices also varied with climate. Among countries, as average temperature increased, so did the frequency of use of chilis, garlic, and onion (Figures 6 and 7), as well as that of anise, cinnamon, coriander, cumin, ginger, lemongrass, turmeric, basil, bay leaf, cardamom, celery, cloves, green peppers, mint, nutmeg, saffron, and oregano (see also Hirasa and Takemasa 1998). The first 10 of these spices are "highly inhibitory" (at least 75% of tested bacterial species inhibited; see Figure 4), and the positive relationships were statistically significant. There were negative relationships between temperature and frequencies of use for 10 other spices, but they were significant only for dill and parsley, neither of which has potent antimicrobial activity.

Prediction 3. A greater proportion of bacteria should be inhibited by recipes from hot climates than from cool climates. In support of this prediction, as average annual temperatures increased among countries, the mean fraction of recipes that called for each one of the highly inhibitory spices used in those countries increased significantly (Figure 8a). However, this correlation did not hold for less inhibitory spices (Figure 8b). There was also a positive relationship between the fraction of bacterial species inhibited by each spice and the fraction of countries that used that spice, indicating widespread use of the spices that are most effective against bacteria.

To further test this corollary, we tried to determine if spices used in each country are particularly effective against local bacteria. Unfortunately, however, no comprehensive lists of indigenous bacteria are available for any country in our sample. To estimate inhibition, therefore, we chose 30 meat-based recipes at random from the cookbooks for each country and tallied how many of 30 "target" bacterial species would be inhibited or killed by at least one spice in each recipe. The target bacteria were those that have been challenged experimentally with the greatest number of spices, including such widespread species as Aeromonas hydrophila, Bacillus cereus, Bacillus subtilus, Clostridium botulinum, Listeria monocytogenes, Escherichia coli, Salmonella pullorum, Staphylococcus aureus, and Streptococcus faecalis. Results of this analysis (Figure 9) showed that as annual temperatures increased, the estimated fraction of food spoilage bacteria inhibited by the spices in each country's recipes increased significantly. Therefore, the cuisine of hotter countries potentially has greater antibacterial activity.

Prediction 4. Within a country, cuisine from high latitudes and elevations (i.e., cooler climates) should contain fewer and less potent spices than cuisine from lower latitudes and elevations. We located regional cookbooks for only two countries, China and the United States. Consistent with the prediction, in both countries the total number of spices used, the fraction of recipes that called for at least one spice, and the frequency of use of highly inhibitory spices were greater in southern regions than in northern regions. The mean number of spices per recipe was greater in southern China than in northern China, but no such difference was evident in the United States (Table 1 ). In both countries, the spices called for in an average southern recipe had significantly greater antibacterial potential than those in northern recipes, mirroring the among-country pattern (Billing and Sherman 1998). Because altitude-specific cookbooks are rare, we were unable to evaluate how altitude affects spice use.

Prediction 5. Quantities of spices called for in recipes should be sufficient to produce antimicrobial effects, and cooking should not destroy the potency of phytochemicals. The primary literature in food microbiology that we surveyed usually reported the minimum concentrations of purified phytochemicals that were necessary to inhibit growth of foodborne bacteria in vitro. Typically, these were solutions containing 0.5-4.0% purified spice chemicals (i.e., 30-2000 pg/ml), which is within the range of spice concentrations used in cooking (Shelef 1984, Giese 1994, Hirasa and Takemasa 1998). However, there are as yet no analyses of how different amounts and types of spices affect microorganisms in cuisine. Evaluating the antimicrobial efficacy of various spices in vivo (i.e., restaurants and homes) would be a fascinating (and potentially lucrative) project.
[Graph]
Figure 3. Figure 4.

Regarding the effects of cooking, most phytochemicals are thermostable, although a few are destroyed by heat (Moyler 1994). Some spices (e.g., garlic, pepper, rosemary, and onion) are typically added at the beginning of cooking, whereas others (e.g., parsley and cilantro [i.e., coriander leaf] ) are added near the end (Figure 10). According to cookbook authors, the "delicate" flavors of the latter would be destroyed by heat. If, as seems likely, thermostable spices are the ones added early and thermolabile spices are added later (or are used primarily as condiments), differences in timing of use may function to maintain beneficial antimicrobial properties (and corresponding flavors) until food is served.

Spice synergism

Pepper and lemon (and lime) juice are among the most frequently used spices (Figure 3), but they are unusual in that the frequency with which they are used does not change much across the temperature gradient (Figure 11). Moreover, they are among the least effective bacteriocides (Figure 4). Do these patterns weaken the antimicrobial hypothesis, or do these two spices function in a different way than "typical" spices?
[Graph]
Figure 5.

We believe that the second explanation is correct, and we suggest that pepper and citric acid play special roles-that is, as synergists. Citric acid potentiates the antibacterial effects of other spices because low pH disrupts bacterial cell membranes (Booth and Kroll 1989). Foods to which lemon or lime juice are added require less heating to cause the same levels of bacterial mortality that take place in foods cooked at higher pH and temperature for a longer time. Black pepper comes from Piper nigrum, an exclusively tropical plant that has several useful properties. For example, the compound piperine inhibits the ubiquitous, deadly bacterium Clostridium botulinum (Nakatani 1994). Black pepper is also a "bioavailability enhancer," meaning that it acts synergistically to increase the rate at which cells, including microorganisms, absorb phytotoxins (Johri and Zutshi 1992).

Many other spices exhibit greater antibacterial potency when they are mixed than when used alone (Ziauddin et al. 1996). Some are combined so frequently that the blends have acquired special names. An intriguing example is the French "quatre epices" (pepper, cloves, ginger, and nutmeg), which is often used to make sausages. Sausages (botulus in Latin) are a rich medium for bacterial growth and have frequently been implicated as the source of botulinum toxin. Other blends, such as curry powder (which contains 22 different spices), pickling spice (15 spices), and chili powder (10 spices), are broadspectrum antimicrobial melanges.

Other uses of spices In addition to their uses in cooking, individual spices and blends are employed as coloring agents, antivirals (including suppressing HIV), brain stimulants, and aphrodisiacs (Hirasa and Takemasa 1998). Among traditional societies, many spice plants also have ethnopharmacological uses, often as topical or ingested antibacterials and vermicides (Chevallier 1996, Cichewicz and Thorpe 1996). A few spices, particularly garlic, ginger, cinnamon, and chilis, have for centuries been used to counteract a broad spectrum of ailments, including dysentery, kidney stones, arthritis, and high blood pressure (Johns 1990, Duke 1994).

However, the use of spices in food preparation differs from medicinal use in three ways. In cooking, spices are used without regard to diners' health status, they are used in tiny quantities, and they are routinely added to specific recipes. This pattern suggests that the "targets" of spice chemicals are on or in the food before it is ingested. By contrast, in medicinal usage, spices are taken in response to particular maladies, in large quantities, and not with any particular dish-more like swallowing a pill than preparing a meal.

An interesting question is whether other animals also "spice" foods. Presently, the answer appears to be "no." However, "vegetation" does form a small but significant fraction of the diet of most wild carnivores, including foxes, coyotes, and cougars (e.g., Parker 1995). Undoubtedly, much of this plant material serves as nutrition, for example, when meat is scarce. Nevertheless, frequent ingestion of vegetation is potentially interesting in the context of the antimicrobial hypothesis because most wild carnivores scavenge carrion, so they are frequently exposed to food-spoilage bacteria and fungi. Moreover, some animals that store food add plants with antibacterial and antifungal properties to their caches (e.g., brown bears sometimes cover carcasses with Spaghnum moss [Elgmork 1982], and some stingless bees build honey pots by mixing plant resins with wax [Roubik 1983]). These possible prophylactic uses should not be confused with consumption of aromatic plants by wild primates as a potential means of "self-medication" (e.g., Huffman and Wrangham 1994).

Costs of spices

In light of the beneficial effects of spices, why aren't spices used equally often everywhere? The answer probably lies in the costs of spice use, including financial costs to procure parts of plants that do not grow locally (e.g., consider the price of Spanish saffron), illnesses caused by ingesting spices that are themselves contaminated (e.g., with bacteria, fungi, or animal feces), and other hazards of ingesting too many plant secondary compounds and essential oils. Indeed, Ames et al. (1990) and Beier and Nigg (1994) reviewed evidence that phytochemicals in many common spices have mutagenic, teratogenic, carcinogenic, or allergenic properties. As one example, in small quantities chilis have antimicrobial and therapeutic effects, but ingestion of large amounts of capsaicin has been associated with necrosis, ulceration, and carcinogenesis (Surh and Lee 1996). The implication is that too much of a good thing can be bad. In hot climates, benefits of avoiding foodborne illnesses and food poisoning apparently outweigh the various costs of spices. But in cool climates, where unrefrigerated foods decay more slowly, benefits of further retarding spoilage may not be worth the costs and risks. Even in countries where spices are heavily used, pre-adolescent children (Rozin 1980) and women in their first trimester of pregnancy (Profet 1992) typically avoid highly spiced foods, especially meats. These differences in spice use may have a similar adaptive basis. For example, Profet (1992) suggested that morning sickness may function to reduce maternal intake of foods containing teratogens during the early phase of embryogenesis, when delicate fetal tissues are most susceptible to chemical disruption. Indeed, women who experience morning sickness are less likely to miscarry than women who do not (Weigel and Weigel 1989). Young children, who are growing rapidly, may also be particularly sensitive tc environmental mutagens. Once pregnancy has progressed into the second trimester and once children reach puberty, the dangers of food poisoning and foodborne illnesses may again outweigh the mutagenic risks associated with phytochemicals (Flaxman and Sherman in press). Interestingly, maternal ingestion of spices late in pregnancy or during lactation can slightly bias offspring toward accepting spices (e.g., Altbacker et al. 1995).
[Photograph]
Figure 6.

Alternative hypotheses to explain spice use

The antimicrobial hypothesis is not the only explanation that has been proposed to explain spice use; however, careful consideration of the alternatives reveals that all have significant flaws. For example, one proximate hypothesis is that spices disguise the smell and taste of spoiled foods (Govindarajan 1985). Our finding that traditional meat-based recipes from hotter countries more frequently called for spices, and more pungent spices, is consistent with this idea because there would more often be foul smells and bad tastes to "cover up" due to rapid spoilage. However, the problem with this hypothesis as an ultimate (evolutionary) explanation is that it ignores the potentially serious negative consequences of ingesting foods laced with bacteria or their toxins. Even poorly nourished individuals would often be better off if they recognized and passed up foods containing potentially deadly spoilage microorganisms.

A second proximate alternative to explain spice use is that spicy foods are preferred in hot climates because they increase perspiration and help cool the body evaporatively. However, although chilis and horseradish can cause sweating in some people, most spices do not have this effect (Rozin and Schiller 1980). Thus, evaporative cooling cannot be a general explanation for the increased spice use in hot climates. Moreover, physiological mechanisms of temperature regulation obviously operate to keep us cool without the necessity of finding, eating, and dealing with the potentially negative side effects of phytochemicals.
[Graph]
Figure

One alternative ultimate hypothesis for spice use is that wherever spices are difficult to obtain and are therefore expensive, individuals signal their wealth and social status (e.g., to rivals or potential mates) by using them lavishly. This hypothesis would apply primarily to spice plants with restricted ranges (e.g., pepper, allspice, fenugreek, nutmeg, and cinnamon). However, it does not predict or explain the multiple positive correlations between temperature and spice use we found for spices that are available ubiquitously (e.g., Figure 7). Also, this hypothesis is difficult to reconcile with the fact that the rarest spices tend to be used most commonly in the tropics, because it is in these locations where the plants are endemic and, presumably, therefore, least expensive.

A second alternative ultimate hypothesis is that spices supply chemicals that, in small quantities, have beneficial effects other than inhibiting food spoilage microorganisms. For example, certain phytochemicals, especially those found in garlic and onions, can aid digestion, modulate energy metabolism, and even help postpone some degenerative diseases, such as diabetes and cancer (Johns and Chapman 1995). Some other phytochemicals, particularly those in cloves, rosemary, sage, pepper, and mace, are powerful antioxidants (Lin 1994, Hirasa and Takemasa 1998). By retarding the oxidation of oil or fat, phytochemicals help preserve foods and also reduce the production of free radicals, which have been linked to cancer and aging. These effects are undeniably important, but they probably do not represent the primary reason for spice use because not all spices have these beneficial properties. Moreover, the need for micronutrients or antioxidants does not predict or explain the use of spices in recipes or the multiple positive correlations between temperature and spice use shown in Figures 5, 7, 8, and 9).

Finally, it is also possible that spice use may not confer any benefits. Under this hypothesis, patterns of spice use arise because people just take advantage of whatever aromatic plants are available to improve the taste of their food. Perhaps the phytochemicals in spices happen to resemble those found in sought-after foods, such as fat and sugar (Rozin and Vollmecke 1986), and as a result spices taste good. If this idea were correct, spice chemicals should be highly palatable, and spice-use patterns should correspond to local availability of spice plants.

However, neither prediction is fully supported. Although some spices are initially appealing (e.g., cinnamon, basil, and thyme), pungent spices, such as garlic, ginger, anise, and chilis, are distasteful to most people, especially children (Rozin 1980). Indeed, the capsaicin receptor is a heat-activated ion channel in the pain pathway (Caterina et al. 1997). For most unpalatable substances, an initial negative response is sufficient to maintain avoidance throughout life. However, preferences for spices develop during individuals' lifetimes, usually under familial guidance. Parents encourage their children to use spices, and most children eventually come to like (or at least accept) them, implying that spice use is beneficial.
[Graph]
Figure 8.
[Graph]
Figure 9.

In addition, spices are not necessarily more available in hot climates than in cool ones. There is no relationship between the number of countries in which each spice plant grows (i.e., its native and domesticated range) and either the number of countries in which it is used or their annual temperatures (Billing and Sherman 1998). Because spices have been cultivated for thousands of years in the Old World (Zohary and Hopf 1994) and hundreds of years in the New World (Coe 1994), it seems likely that these patterns of spice plant availability reflect those that occurred when traditional recipes were developing.

Thus, correlations between spice use and annual temperature must be due to people in hot countries using a larger proportion of whatever spices are available locally (or importing more spices). Indeed, for 22 of 30 spices ( 73 % ), a larger percentage of recipes called for the spice in countries where the plant grows than where it does not grow; for 14 of the spices, these differences were significant (P < 0.05, Mann-Whitney tests). Of course, the spice trade (Figure 2) facilitates the use of nonindigenous spices. For example, onion and pepper are the two most frequently used spices in the world (Figure 3). Allium grows in all 36 countries we examined, but Piper grows in only 9 countries. Pepper is the world's most frequently traded spice (more than 90 million pounds per year are imported into the United States alone; Tainter and Grenis 1993). Thus, although local availability certainly influences spice use, use is not dictated solely by local availability.

Origins of spice use

How did spice use begin? We hypothesize that people may have begun cooking with spices whose flavors were initially appealing or that made them feel good (due to digestive or vermicidal effects, among other things). As a result, spice-using families may also have been less likely to suffer from foodborne illnesses or food poisoning than families that did not use spices, especially in hot climates. Furthermore, spice-using families probably would have been able to store foods longer before they spoiled, enabling them to tolerate prolonged periods of food scarcity. Observation and imitation of the food-preparation habits of these healthier families by neighbors could have spread spice use rapidly through a society. Families that used appropriate spices would presumably rear more healthy offspring, who would then learn spice-use traditions from their parents. It even seems possible that people who lived in areas where certain spices were traditionally used might have developed physiologically heightened abilities to taste those phytochemicals. The possible existence of such intergroup variations in taste receptor sensitivity to spices are just beginning to be explored (Drewnowski and Rock 1995).
[Photograph]
Figure 10. '

Eventually, however, new foodborne bacteria or fungi would immigrate, or indigenous microorganisms would evolve resistance to local spices. Individuals eating foods contaminated by these microbes would become ill. After humans, like many other creatures, eat something that makes them sick, they tend to avoid that taste (Milgram et al. 1977, Pelchat and Rozin 1982). The adaptive value of such "taste-aversion learning" is obvious (Rozin and Vollmecke 1986, Letarte et al. 1997). Adding a different spice to a food that caused such an illness might change its flavor enough to make it palatable againbecause it tastes like a new food. At the same time, if the spice were to kill the microorganism(s) that caused the illness in the first place, then the food would again be rendered safe for consumption. As a result of this sequence of events, food aversions would more often be associated with unspiced (and unsafe) foods, whereas food likings would be associated with spicy dishes, especially in climates where foods spoil rapidly. Over time, the number of spices per recipe would proliferate due to iteration of this process-that is, sequential changes in taste, associated with inhibiting different bacteria and fungi.

Antimicrobial value of spices today

Despite the widespread availability of electrical refrigeration, antimicrobial properties of spices may still be useful. For example, there is an order-of-magnitude difference in the frequency of foodborne illnesses between modern Japan and Korea, nearby countries with similar temperate climates. During 1971-1990, food poisoning-primarily of bacterial origin-affected 29.2 out of every 100,000 Japanese but only 3.0 out of every 100,000 Koreans (Lee et al. 1996). Lee et al. (1996) suggested that the difference may have been due to cultural variations in food handling and preparation, and this explanation may well be correct. But, in addition, Korean meat-based recipes are spicier than those of Japan. Although meat-based recipes of .Japan collectively used more kinds of spices (14) than those of Korea (8), Korean recipes more frequently called for at least one spice, contained more spices per recipe (Table 1), and more frequently called for highly inhibitory spices (Billing and Sherman 1998). As a result, an average Korean recipe most likely inhibits a significantly greater fraction of bacteria than an average Japanese recipe. One possible explanation for the fact that traditional Japanese recipes do not call for more spices is that they date from times when fresh seafood was continuously available from local waters. Today, more food is imported, and it comes from farther away. Traditional Japanese recipes may simply not include enough spices (antimicrobials) to cope with the pathogens in the imported food supply.

Of course, spice use is not the only way in which humans attempt to hold foodborne pathogens at bay. Meat products have traditionally been preserved by thoroughly cooking, smoking, drying, and salting them. Indeed, salt, which is available the world over, has been used for preservation for centuries (Multhauf 1996). And today, of course, the "front line" of defense against spoilage is refrigeration and freezing. We hypothesize that all these practices have been adopted for essentially the same reason: to minimize the impact of microorganisms that colonize our food.

Conclusion

Use of spices takes advantage of plant defensive compounds. Not surprisingly, in view of their evolved functions, these phytochemicals have antioxidant, antimicrobial, and antiviral properties. The use of spices essentially borrows plants' recipes for survival and puts them to similar use in cooking. Over time, recipes should "evolve" as new bacteria and fungi appear or indigenous species develop resistance to phytochemicals, requiring the addition of more spices or new spices to combat them effectively. However, there is a limit to how much of any one spice can be added before beneficial phytochemicals become phytotoxins. Thus, cookbooks from different eras are more than just curiosities. Essentially, they represent written records of our coevolutionary races against foodborne diseases. By cleansing foods of pathogens before consumption, spice users contribute to the health, longevity, and fitness of themselves, their families, and their guests. A Darwinian view of gastronomy thus helps us understand why "some like it hot" (spicy, that is!).

Acknowledgments

We thank John Alcock, Thomas A. Gavin, Thomas Neuhaus, H. Kern Reeve, Laurel Southard, and Cynthia Kagarise-Sherman for ideas and encouragement; Lee A. Dugatkin, Thomas Eisner, Paul W. Ewald, Rebecca Chasan, Gail Jarrow, Mary Ann Shallenberger, Philip S. Sherman, and an anonymous reviewer for suggestions on the manuscript; the librarians at Cornell University's Mann and Nestle Libraries for assistance with references; and the Howard Hughes Medical Institute, the National Science Foundation, and the College of Agriculture and Life Sciences at Cornell University for financial support.
[Graph]
Figure 11.
[Reference]
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Beier RC, Nigg HN.1994. Toxicology of naturally occurring chemicals in food. Pages 1186 in Hui YH, Gorham JR, Murrell KD, Cliver DO, eds. Foodborne Disease Handbook. Vol. 3: Diseases Caused by Hazardous Substances. New York: Marcel Dekker.
Beuchat LR.1994. Antimicrobial properties of spices and their essential oils. Pages 167179 in Dillon VM, Board RG, eds. Natural Antimicrobial Systems and Food Preservation. Wallingford (UK): CAB International.
Billing J, Sherman PW. 1998. Antimicrobial functions of spices: Why some like it hot. Quarterly Review of Biology 73: 3-49.
Booth IR, Kroll RG.1989. The preservation of foods by low pH. Pages 119-160 in Gould GW, ed. Mechanisms of Action of Food Preservation Procedures. London: Elsevier.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. 1997. The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389: 816-824.
Chevallier A. 1996. The Encyclopedia of Medicinal Plants. New York: DK Publishing. Cichewicz RH, Thorpe PA. 1996. The antimicrobial properties of chile peppers (Capsicum species) and their use in Mayan medicine. Journal of Ethnopharmacology 52: 61-70.

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Coe SD. 1994. America's First Cuisines. Austin (TX): University of Texas Press. Deans SG, Ritchie G. 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5: 165-180. Dillon VM, Board RG, eds. 1994. Natural Antimicrobial Systems and Food Preservation. Wallingford (UK): CAB International. Drewnowski A, Rock CL. 1995. The influence of genetic taste markers on food acceptance. American Journal of Clinical Nutrition F 7 SnF,-11

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Duke JA.1994. Biologically active compounds in important spices. Pages 201-223 in Charalambous G, ed. Spices, Herbs, and Edible Fungi. Amsterdam: Elsevier. Elgmork K. 1982. Caching behavior of brown bears (Ursus arctos). Journal of Mammalogy 63: 607-612.
Farrell KT.1990. Spices, Condiments, and Seasonings. 2nd ed. New York: Van Nostrand Reinhold.
Flaxman SM, Sherman PW. In press. Morning sickness: A mechanism for protecting the embryo. Quarterly Review of Biology. Fraenkel GS.1959. The raison d'etre of secondary plant substances. Science 129: 14661470.
Giese J. 1994. Spice and seasoning blends: A taste for all seasons. Food Technology 48: 87-90.

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Govindarajan VS. 1985. Capsicum production, technology, chemistry, and quality. Part 1: History, botany, cultivation, and primary processing. CRC Critical Reviews in Food Science and Nutrition 22:109-176. Hargreaves LL, Jarvis B, Rawlinson AP, Wood JM. 1975. The antimicrobial effects of spices,

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herbs and extracts from these and other food plants. Scientific and Technical Surveys, British Food Manufacturing Industries Research Association 88: 1-56. Hirasa K, Takemasa M. 1998. Spice Science
and Technology. New York: Marcel Dekker. Hobbs BC, Roberts D. 1993. Food Poisoning and Food Hygiene. 6th ed. London: Edward Arnold.

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Huffman MA, Wrangham RW. 1994. Diversity of medicinal plant use by chimpanzees in the wild. Pages 129-148 in Wrangham RW, McGrew WC, DeWaal FBM, Heltne PG, eds. Chimpanzee Cultures. Cambridge (MA): Harvard University Press. Hui YH, Gorham JR, Murrell KD, Cliver DO, eds. 1994. Foodborne Disease Handbook. Vol. 1: Diseases Caused by Bacteria. New York: Marcel Dekker.
Johns T. 1990. With Bitter Herbs They Shall Eat It: Chemical Ecology and the Origins of Human Diet and Medicine. Tucson (AZ): University of Arizona Press. Johns T, Chapman L. 1995. Phytochemicals ingested in traditional diets and medicines as modulators of energy metabolism. Recent Advances in Phytochemistry 29: 161188.

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Johri RK, Zutshi U. 1992. An Ayurvedic formulation "Trikatu" and its constituents. Journal of Ethnopharmacology 37: 85-91. Lee W-C, Sakai T, Lee M-J, Hamakawa M, Lee S-M, Lee I-M. 1996. An epidemiological study of food poisoning in Korea and Japan. International Journal of Food Microbiology 29: 141-148.
Letarte A, Dube L, Troche V.1997. Similarities and differences in affective and cognitive origins of food likings and dislikes. Appetite 28: 115-129.
Lin RI-S. 1994. Pharmacological properties and medicinal use of pepper (Piper nigrum L.). Pages 469-481 in Charalambous G, ed. Spices, Herbs, and Edible Fungi. Amsterdam: Elsevier.

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Milgram NW, Krames L, Alloway TM. 1977. Food Aversion Learning. New York: Plenum Press.
Moyler DA. 1994. Spices-recent advances. Pages 1-71 in Charalambous G, ed. Spices Herbs, and Edible Fungi. Amsterdam: Elsevier.
Multhauf RP. 1996. Neptune's Gift: A History of Common Salt. Baltimore: Johns Hopkins University Press.
Nakatani N. 1994. Antioxidative and antimicrobial constituents of herbs and spices. Pages 251-272 in Charalambous G, ed. Spices, Herbs, and Edible Fungi. Amsterdam: Elsevier.
Parker G. 1995. Eastern Coyote: The Story of Its Success. Halifax (Canada): Nimbus Publishing.

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Parry JW. 1953. The Story of Spices. New York: Chemical Publishers.
Pelchat ML, Rozin P. 1982. The special role of nausea in the acquisition of food dislikes by humans. Appetite 3: 341-351. Profet M. 1992. Pregnancy sickness as adaptation: A deterrent to maternal ingestion of

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teratogens. Pages 327-365 in Barkow J, Cosmides L, Tooby J, eds. The Adapted Mind. New York: Oxford University Press. Roubik DW. 1983. Nest and colony characteristics of stingless bees from Panama (Hymenoptera: Apidae). Journal of the Kansas Entomological Society 56: 327-355. Rozin P. 1980. Acquisition of food preferences and attitudes to food. International Journal of Obesity 4: 356-363.
Rozin P, Schiller D. 1980. The nature and acquisition of a preference for chili pepper by humans. Motivation and Emotion 4: 77100.

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Rozin P, Vollmecke TA. 1986. Food likes and dislikes. Annual Review of Nutrition 6: 433-456.

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Ruhlen M. 1987. A Guide to the World's Languages. Vol.1: Classification. Stanford (CA): Stanford University Press.
Scheiper R.1993. Hot Spice. Contact 57. Springfield (NJ): Haarman and Reimer Shelef LA.1984. Antimicrobial effects of spices.
Journal of Food Safety 6: 29-44. Sherman PW. 1988. The levels of analysis. Animal Behavior 36: 616-618. Sockett PN.1995. The epidemiology and costs of diseases of public health significance, in relation to meat and meat products. Journal of Food Safety 15: 91-112.
Surh Y-J, Lee SS. 1996. Capsaicin in hot chili pepper: Carcinogen, co-carcinogen, or anticarcinogen? Food and Chemical Toxicology 34: 313-316.

[Reference]
Tainter DR, Grenis AT. 1993. Spices and Seasonings. New York: VCH Publishers. Todd ECD. 1994. Surveillance of foodborne disease. Pages 461-536 in Hui YH, Gorham JR, Murrell KD, Cliver DO, eds. Foodborne Disease Handbook. Vol.1: Diseases Caused by Bacteria. New York: Marcel Dekker. Varnam AH, Evans MG. 1991. Foodborne Pathogens. London: Mosby-Year Book. Walker JRL. 1994. Antimicrobial compounds in food plants. Pages 181-204 in Dillon VM, Board RG, eds. Natural Antimicrobial Systems and Food Preservation. Wallingford (UK): CAB International. Weigel RM, Weigel MM. 1989. Nausea and vomiting of early pregnancy and pregnancy outcome: A meta-analytical review. British Journal of Obstetrics and Gynaecology 96: 1312-1318.
[WHO] World Health Organization. World Health Report 1996. The State of World Health. Geneva: World Health Organization.

[Reference]
Zaika LL. 1988. Spices and herbs: their antimicrobial activity and its determination. Journal of Food Safety 9: 97-118. Ziauddin KS, Rao HS, Fairoze N. 1996. Effect of organic acids and spices on quality and shelf-life of meats at ambient temperature. Journal of Food Science and Technology 33: 255-258.
Zohary D, Hopf M. 1994. Domestication of Plants in the Old World: The Origin and Spread of Cultivated Plants in West Asia, Europe, and the Nile Valley. 2nd ed. Oxford (UK): Oxford University Press.

[Author note]
Paul W. Sherman is a professor at Cornell University. He studies the behavioral ecology of various birds and mammals and teaches animal behavior and Darwinian medicine. Jennifer Billing was an undergraduate Honors student at Cornell when she began studying spices. She now teaches biology and chemistry at The Dalton School in New York City. 1999 American Institute of Biological Sciences.

Reproduced with permission of the copyright owner. Further reproduction or distribution is prohibited without permission.

Life Enhancement:: Goodbye to Antibacterial-Resistant Bacteria - Nov. 1999

2005-06-24T02:52:00.000-07:00

Life Enhancement:: Goodbye to Antibacterial-Resistant Bacteria - Nov. 1999

New & Improved Bye-Lori
Goodbye to Antibiotic-Resistant Bacteria
Mastic Plus New Phytonutrients
for Improved Gastrointestinal Health

by Will Block

oodborne illnesses affect tens of millions of Americans each year, according to the Centers for Disease Control (CDC). The consequences are grim, with as many as 5000 deaths, 325,000 hospitalizations, and 76 million cases of gastrointestinal illness reported each year in the United States.1 In what officials call a new picture of the devastation of foodborne disease, the CDC has found fewer deaths than previously estimated, but far more illnesses. Each year, approximately one out of every four Americans falls prey to a foodborne illness.

Foodborne illnesses are not new to the world. In fact, they have been the rule rather than the exception throughout history. There are many variables contributing to food contamination, including the safety of the water supply, waste disposal, and personal hygiene. The overriding problem is the evolution of drug-resistant bacteria that are immune to medicine's best antibiotics. More virulent populations of bacteria make infections more difficult to treat, requiring newer, stronger antibiotics - and so the cycle keeps repeating.

Figure 1.Overwhelming all defenses. Percentage of hospital-acquired enterococcal bacteria reported as resistant to vancomycin (the antibiotic of last resort). Enterococcus faecium is the third leading cause of bloodstream infections that are now running rampant in hospitals, owing to antibiotic resistance.
Antibiotic resistance is growing, warn medical experts who have seen the writing on the wall.1 Alarm is growing at hospitals across the nation, where the potency of even the antibiotic of last choice, vancomycin, has been greatly diminished (see Figure 1). Many antibiotics commonly prescribed for respiratory tract infections are losing their effectiveness, according to researchers who have been tracking the medical statistics. Dr. Ronald Jones, a leading investigator in a program called Sentry, at the University of Iowa College of Medicine, Iowa City, is alarmed that antibiotic resistance of the organism Pneumococcus, a common cause of respiratory tract infections, has jumped from 4% to nearly 40% in less than 20 years.

Jones is urging caution in the use of antibiotics, for patients as well as doctors. Many patients insist on antibiotics when they aren't needed or would not even be effective, such as for a viral infection, and too many doctors go along, even though they should know better. It is estimated that up to 50% of all antibiotics prescribed are for conditions for which they have no value. About 2/3 of all complaints in a doctor's office relate to the respiratory tract, and this, according to Jones, is where resistance is growing most rapidly. He has appealed for an emergency approach that focuses on education, surveillance, and new research. Just 11 years ago, e.g., ciprofloxacin was effective against most major pathogens. Now more than 10% of organisms are resistant. One of the problems with some of the older antibiotics is that they promote mutations in microorganisms, making them stronger, so resistance develops quickly.

It is interesting, but predictable, that in countries where antibiotics are available over the counter, resistance is even higher than in the United States. In Mexico, for example, bacterial resistance to penicillin is 60%, vs. only 35% north of the border.

REGIONAL SOLUTIONS TO BACTERIALLY SPREAD DISEASE
One possible solution to the problem is to use more spices, for their antibacterial properties. Throughout history, spices have been used in food preparation worldwide, although their use has differed considerably among cultures and countries. Two researchers, Billing and Sherman, analyzed the data on the frequency of use of 43 spices in the meat-based cuisines of the 36 countries for which they could locate traditional cookbooks.3 They also looked at the temperature and precipitation statistics in each country, the range of available spice plants, and the antibacterial properties of each spice.

The researchers found that many spices do inhibit or kill microorganisms that cause food spoilage and that there was a positive correlation between the use of spices in food preparation and average annual temperature. The traditional view - that spices provide no medical benefits, but are used only to provide macronutrients, to disguise the taste and smell of spoiled foods, or increase perspiration and thus evaporative cooling - was not supported by the evidence. Billing and Sherman believed that the ultimate reason for spice use was, in fact, to help cleanse foods of pathogens and thereby to contribute to the health, longevity, and reproductive success of people who find their flavors enjoyable. When they tallied the amount of spices used in the typical recipe, 0.25 to 3 grams per kilogram of food, they found that it tended to fall within a range "sufficient to yield useful antibacterial effects."

SPICES MAKE FOOD SAFER
The food-poisoning rates in Japan and Korea are different. In Japan, there were 30 incidents of food poisoning for every 100,000 diners between 1971 and 1990. In Korea during the same period, the incidence was only 3 out of 100,000, or 10% of Japan's rate. Although the two countries have similar climates, Koreans eat their food spicier. In fact, for every two potent antimicrobial spices in Japanese recipes, there are three in Korean recipes, or 50% more. These data seem to bear out the argument made by Billing and Sherman, who state, "The estimated proportion of foodborne bacteria inhibited by an average recipe is significantly higher in Korean (51%) than Japanese cuisine (12%)."

THE NEW WORLD OF SPICES
The discovery of America was the accidental result of the search for spices. Christopher Columbus was quite knowledgeable about medicines, since he supplemented his readings with trips to famous regional pharmacies.4 Having visited the Greek archipelago, including the Isle of Chios, he knew that the concept of disease-preventing spices encompassed more than just the flowers and seeds of small plants. It could also entail the roots, leaves, bark, and even the sap, of trees. One tree, in particular, had been cultivated to produce a highly antibacterial sap that was believed to be more curative and thus more valuable than all the other "spices," and that was the mastic tree, Pistacia lentiscus.

As proof of his belief in mastic sap, Columbus wrote several letters upon his return from his first voyage to America, one to Luis De Sant Angel, Treasurer of Aragon, announcing his discovery of mastic in the New World:

To speak, in conclusion, only of what has been done during this hurried voyage, their Highnesses [Ferdinand and Isabella of Spain] will see that I can give them as much gold as they desire, if they will give me a little assistance, spices, cotton, as much as their Highnesses may command to be shipped, and mastic as much as their Highnesses choose to send for, which until now has only been found in Greece, in the isle of Chios, and the Signoria can get her own price for it.5

THE POWER OF MASTIC
Columbus thought, as presumably many botanical pharmacists did, that mastic could cure cholera, and other ailments as well. So to encourage sightings after landing in the New World, he offered a reward to the sailor who first located mastic. What Columbus found, however, was not mastic - to this day it is cultivated successfully only on Chios - but gumbo-limbo, or turpentine tree (Bursera simaruba). Columbus was told (in sign language) by the natives that gumbo-limbo - which exudes a resin that bears a striking resemblance to the real mastic sap and, like it, has a turpentine-like taste - was "good for a stomach ache." Since mastic was beneficial for the gastrointestinal tract, it is no wonder that in his rush to identify "mastic," he mistook gumbo-limbo for the real thing.

In the centuries before and after Columbus, so powerful was the mystique of mastic (also know as mastiche) that some believed it could even cure the plague (see Discovering Antibacterial Mastic - Page 1 - April 1999, Explorer Columbus - April 1999, and Goodbye Pylori - March 1999). In an article in the journal Lloydia, J. L. Hartwell cites hundreds of historical references, including materia medicas, natural histories, medical treatises, medieval pharmacopoeias, clinical reports, and Egyptian medical papyri, on the wide usage of mastic over the ages for gastrointestinal ulcers, diarrhea, and a wide variety of other medical problems.7

In retrospect, many pharmacognosists have concluded that the claims about mastic were fabrications, representing a search for the Holy Grail of medications. Yet such is our current understanding of the science underlying mastic that it now appears that the "exaggerations" about mastic's miraculous qualities were probably closer to the mark. Their error was only about the diseases that it could cure, not about its power.

BYE-LORI MEANS WHAT IT SAYS - GOODBYE, H. PYLORI
Bye-Lori, Life Enhancement's gastrointestinal support product, is revolutionary, because it is the first identified natural product that can deliver a serious blow to the armor of what is undoubtedly one of the most feared gastrointestinal bacteria in the world, Helicobacter pylori. It has been estimated that about 40% of the entire population of the planet is infected with H. pylori. This means that about 2.4 billion people on earth would probably lead healthier lives if H. pylori were eradicated from their stomachs.

Because H. pylori has been intimately connected with peptic, gastric, and even duodenal ulcers, as well as carcinomas of the stomach, the widespread use of a mastic-containing product such as Bye-Lori would be a boon to human health worldwide.

In a double-blind, placebo-controlled trial involving 38 human subjects with duodenal ulcers, 1 gram of mastic powder was given once per day before breakfast to 20 subjects for a period of two weeks, while a 1-gram lactose placebo was given to the other 18 subjects.7 A high level of symptomatic relief was reported in 80% (16 subjects) in the mastic group, and when their stomachs were examined with a viewing scope, 70% (14 subjects) were proven to have significant healing. The site of the original ulcer had been completely replaced by epithelial tissue (the cells that normally line the gastrointestinal tract), without any appearance of new ulcers. According to the authors, the differences attributed to mastic were highly significant. There were no side effects reported in this study.

In another study, five male patients and one female patient with benign gastric ulcers who had not been treated for ulcers within two months received 2 grams per day of mastic powder, 1 gram in the morning before breakfast and 1 gram at bedtime, for four weeks.8 Complete symptomatic relief was found in all six subjects, including one man with a double gastric ulcer. When the sites of the ulcers were viewed, five of the six subjects, including the double-ulcer man, had completely new epithelial cell growth over the ulcer at the end of the four weeks. No side effects were reported. The authors noted that they had previously documented another 14 cases of gastric ulcers treated successfully with mastic, as confirmed by upper gastrointestinal viewing during a one-year period of treatment and follow-up.9 The amount of mastic used in this study did not exceed the quantities used by the general public chewing mastic gum.

INTRODUCING NEW & IMPROVED BYE-LORI
With the enormous success of Bye-Lori, we wanted to create an even more advanced formulation, to provide a natural alternative for individuals dealing with difficult or intractable gastrointestinal problems. So we looked at the data for a wide variety of plants that possess antibacterial properties.

THYME AND CINNAMON EFFECTIVE AGAINST H. PYLORI
When the extracts of several plants were examined for their effect on H. pylori, cinnamon and thyme were found to be effective.10 Both extracts, but particularly thyme, had a significant ability to inhibit H. pylori growth and limit its urease activity. Urease, an enzyme that breaks urea down into carbon dioxide and ammonia, is an important virulence factor for H. pylori and is critical for bacterial colonization of the human gastric mucosa. The amount of thyme needed to inhibit H. pylori completely was readily achievable with either powdered thyme or a liquid extract.

HYPERFORIN IS EFFECTIVE AGAINST ANTIBIOTIC-RESISTANT BACTERIA
St. John's wort, an herb used for antidepressant purposes,* contains an ingredient, hyperforin, that has been found to possess antibacterial properties. This was shown as far back as 1959 and 1971 by Russian researchers.11,12 A study performed in Romania in 1988 demonstrated a specific antibacterial role for an extract from St. John's wort, a gastrointestinal effect. When the extract was given to rats with aspirin-induced ulcers, a protective, anti-ulcer effect was displayed.13

* Curiously, hyperforin seems to invoke conditions necessary for Hebb's rule (see The Genetic Leap to Greater Memory - November 1999 and Kaehler ST, Sinner C, Chatterjee SS, Philippu A. Hyperforin enhances the extracellular concentrations of catecholamines, serotonin and glutamate in the rat locus coeruleus. Neurosci Lett 1999 Mar 12;262(3):199-202). Thus, it may be regarded as a "speed association" phytonutrient, in addition to its antibiotic role. By containing hyperforin, Bye-Lori now "speaks" to the brain, as well as the stomach.

The chemical structure of hyperforin is unlike that of any other known antibiotic. Even in low concentrations in laboratory studies, hyperforin is effective against a wide range of bacteria, including E. coli and other Gram-positive bacteria, and multiresistant bacteria.14 It is capable of inhibiting penicillin-resistant and methicillin-resistant Staphylococcus aureus. Toxicity levels are low.

A bioavailability study showed that a 300-mg oral administration of St. John's wort containing 5% hyperforin was well tolerated and resulted in serum levels sufficient to support the systemic antibiotic use of hyperforin.16

OVERWHELMING THE OFFENSE
You need only open the pages of your newspaper, turn on the tube, or click onto the Web to hear about virulent bacteria breaking through the best defense lines thrown up by conventional medicine. Not that antibiotics are not of great value - that's not in question. The real question is, are we diminishing their value by overusing them and allowing bacteria to mutate, reprogramming themselves to do us severe harm and even threaten our lives?

A good defense is a good offense, and Bye-Lori - now including hyperforin, thyme, and cinnamon, along with the strength of classic mastic powder - is here. In these days of bacterial consciousness, Bye-Lori could provide the natural, healthy alternative you're looking for.

References

1. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. Food-related illness and death in the United States. Emerg Infect Dis 1999 Sep-Oct;5(5) (in press). http://www.cdc.gov/ncidod/EID/vol5no5/mead.htm#5
2. Zoeller J. Antibiotic Resistance Is Growing, Researchers Report 1999 Sept 17. Tribune Medical Web: http://www.medtrib.com
3. Billing J, Sherman PW. Antimicrobial functions of spices: why some like it hot. Q Rev Biol 1998 Mar;73(1):3-49.
4. Griffenhagen G. Materia medica of Columbus. Int Pharm J 1990;4:271-2.
5. Hart and Channing. American History Leaflets CD Sourcebook of American History 1995 Compact University.
6. Hartwell JL. Plants used against cancer. Lloydia 1967:30/4;379-436.
7. Al-Habbal MJ, Al-Habbal Z, Huwez FU. A double-blind controlled clinical trial of mastic and placebo in the treatment of duodenal ulcer. J Clin Exp Pharm Physiol 1984;11:541-4.
8. Huwez FU, Al-Habbal MJ. Mastic in treatment of benign gastric ulcers. Gastroenterol Japon 1986;21:273-4.
9. Al Habbal MJ, et al. Upper G.I.T. endoscopy in Arbil. Iraq Med J 1982;29:25.
10. Tabak M, Armon R, Potasman I, Neeman I. In vitro inhibition of Helicobacter pylori by extracts of thyme. J Appl Bacteriol 1996 Jun;80(6):667-72.
11. Derbentseva NA, Rabinovych AS, Aizenman BIu, Zelepukha SI, Mandryk TP, Shvaiger MO. Antimicrobial substances from Hypericum perforatum. Mikrobiol Zh (Kiev) 1959;21(5):52-7.
12. Gurevich AI, Dobrynin VN, Kolosov MN, Popravko SA, Riabova ID. Antibiotic hyperforin from Hypericum perforatum L. Antibiotiki 1971 Jun;16(6):510-3.
13. Hriscu A, Stanescu U, Ionescu A, Verbuta A, Gusetoaia F. Study of gastro-protective effects of extractive fractions from Hyperici herba in experimental ulcers of rats. Farmacia (Bucharest); 1988;6:43-50.
14. Schempp CM, Pelz K, Wittmer A, Schopf E, Simon JC. Antibacterial activity of hyperforin from St. John's wort, against multiresistant Staphylococcus aureus and gram-positive bacteria. Lancet 1999 Jun 19;353(9170):2129.
15. Biber A, Fischer H, Romer A, Chatterjee SS. Oral bioavailability of hyperforin from Hypericum extracts in rats and human volunteers. Pharmacopsychiatry 1998 Jun;31 Suppl 1:36-43.

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Indian Spices

2005-06-24T02:48:00.000-07:00

Indian Spices

Indian Spices -Tastes of Paradise

By

V.Krishna Moorthy,Bhaskar Karnick

Contents

01. Introduction

02. Importance of spices

03. Usage

04. India and Spices

05. History of Spices

06. Sources of Spices
07. Culinary Herbs

08. Spices as Aphrodisiac
09. Perfumes and Incenses

10. Spices as Medicine

11. Anti-microbial Functions of Spices
12. Business of spices
13. The Indian share

14. Spices Time Line
15. Glossary of Common Indian Spices
16. Common Indian Spices and Description
17. Taste and Hotness of Spices
18. Links to Spice Resources

1. Introduction

Spice, aromatic vegetable product used as a flavoring or condiment, normally refers to the derivatives from certain herbs like Seeds, Leaves, Bark, roots etc They are used mainly for enhancing taste of the food. The name spice is derived from the word species, which was applied to groups of exotic foodstuffs in the Middle Ages.

Spice term was formerly applied also to pungent or aromatic foods, to ingredients of incense or perfume and to embalming agents. Modern usage tends to limit the term to flavorings used in food or drinks, although many spices have additional commercial uses, e.g., as ingredients of medicines, perfumes, incense, and soaps. Aromatically scented herbal products have been used since ancient times to flavor food and for preparing incenses and perfumes.

The earliest literary record in India on spices is the Rig Veda (around 6000 BC), and the other three Vedas - Yajur, Sama and Atharva. The Rig Veda, one of the ancient Hindu scriptures, lists more than a thousand healing plants. The story of Indian Spices dates back to 7000 years into the past. In the modern world, major trade is related to eating and spices provide the major thrust - traditionally a country of agriculture, India leads the trade.

The very word "Spice" kindles the taste buds and brings pleasure to the mind. A well equipped Indian kitchen has all major verities of Spices stocked. In India, Spices are available in almost all grocery shops. The common Spices which are used in their raw and fresh forms are available in vegetable shops. Spices provide a rich source of home remedies. Some of the daily used Spices have been taken for granted as part of daily food item and are used routinely without a second thought. Even with the giant progress in science, Spices have no replacement, in fact they form the basis of many medical research. Spices are packed in convenient forms - either as powder, liquid, coarsely ground or just farm fresh. Herbs are extensively used as medicine, preservative, perfume etc.

Spices trade is a big business from time immemorial. Spices from India and Far Eastern Asia were in demand from ancient times; they were carried by caravan across China and India to ports of the Mediterranean Sea or the Persian Gulf and thence to the marketplaces of Athens, Rome, and other cities, where they were sold at exorbitant prices.

When overland trade routes from Asia were cut off by the Mongols and Turks, the European demand for spices was a major factor in motivating a search for new trade routes around Africa and across the Atlantic and Pacific oceans. The high price obtainable for spices was partially responsible for the bitter rivalry of European powers for the control of spice-producing areas and of trade routes. Even after adequate supplies of spices were found and means of transportation made available, the cost, long remained very high in Europe and in America. This was largely because of the transportation costs, expenses incident to attempts to retain monopoly of markets and to deliberately limit crops in order to secure high prices.

2. Importance of spices

The spice trade is very ancient. To understand the amazing prestige of spices in ancient times we must remember for one thing that in ancient time food was neither good nor palatable. There were no potatoes; no corn, tea, coffee or chocolate. There were no lemons with which to prepare refreshingly acid beverages, and neither was there sugar with which to sweeten them. However, a dash of pepper, a little cinnamon or ginger, mixed with even the coarsest dishes, could make them palatable.

The earliest trading caravans known in the human history carried spices. The early civilizations of the Mediterranean craved for the spices from India and the other Eastern lands. The Egyptians used herbs and spices in their daily activities. The Egyptian spice expeditions to east coast of Africa are recorded as early 2000 BC. The Roman started sailing to India from Egypt in the first century AD. Stars were the only navigation system available to these early spice traders. A round trip to India took as long as five years. When Europeans were living in a relatively underdeveloped atmosphere, a thriving commerce between East and West flourished through out Indian Ocean and Asia. However, that was destined to change with the introduction of steamed ships.

The demand for spices spread like a wave over Europe - even beyond the fringes of civilization. Alaric the Visigoth demanded as ransom, , when he lay siege to Rome, 3000 pounds of pepper Later he demanded, an additional quota of 300 pounds a year. In a short period of history People of Europe, quickly learnt that spices can make their food tastier and can keep their meat fresher. This led to many wars lost and won.

During the Middle Ages in Europe, a pound of ginger was worth the price of a sheep; a pound of mace would buy three sheep or half cow; cloves cost the equivalent of about $20 a pound. Pepper, always fetched greatest price. Pepper was counted out corn by corn. The guards on London docks even down to Elizabethan times, had to have their pockets sewn up to make sure they didn't steal any spices. In the 11th Century, many towns kept their accounts in pepper; taxes and rents were assessed and paid in this spice and a sack of pepper was worth a man's whole life.

3. Usage

Spices can improve the palatability and the appeal of dull diets or spoiled food. Piquant flavors stimulate salivation and promote digestion. Pungent spices can cause sweating, which may even cause a cooling sensation in tropical climates; on the other hand they can add a sense of inner warmth when present in cooked foods used in cold climates. Local and inexpensive herbs and flavors, such as garlic, onion and horseradish, sufficed for the poorer people of old Europe, but influential, rich hosts would wish to impress or politically intimidate their guests with the liberal use of rare exotic spices. These expensive imports could be added in large amounts and in complex mixtures to each course and to accompanying alcoholic beverages to provide a gustatory statement about the wealth, power and initiative of the host. Thus, spices served to make a political statement when a baronial lord invited possible rivals to an expensive display of profligacy at a sumptuous banquet.

On the contrary in India, traditionally, spices formed a part of common man's daily food.

Cultures are defined as much by their foods, especially, Spices - as by music, art, and dress. Selection of spices reflected different ethnic cultures. Active ingredient culturally associated are:

Curry (mixture of turmeric, cumin, coriander, pepper)
Red pepper (capsaicin)
Jalapeno (capsaicin)
Garlic (allicin)
Cinnamon (cinnamic aldehyde)
Oregano (thymol oil)

In the medieval period, there were many reasons for the rise in spice usage and their trade. Primarily , the monotony of a lifetime consuming of bread and gruel resulted in a powerful desire to, literally, spice up the food. Even today it is people in India and other poorest countries of the Third World who are most likely to use spices in their food. Secondly, there was the need for the emerging new class of bourgeoisie to culturally demonstrate power and superiority by purchasing luxury items like spices, used in foods, medicines, and ointments. Additionally, there was a insatiable desire for gold and silver among the Mediterranean’s trading partners in the East viz. the Indians and the Chinese.

4. India and Spices

Indian Spices paid important role in the history of various lands, discovered or destroyed, kingdoms built or brought down, wars won or lost, treaties signed or flouted, favours sought or offered. Spices have also played a political role in the history. The use of spices from the East became a status symbol by the year 1200 and the European preoccupation with the world of spice was born. The use of spice in food meant money and power, and the desire to acquire these precious status symbols led to world exploration pan-global communication, trade, alliances and wars.

Indian Spices also fitted into philosophic concepts of improving health, since it was understood that they could affect the four humors (blood, phlegm, yellow bile and black bile) and influence the corresponding moods (sanguine, phlegmatic, choleric and melancholic). Thus, ginger would be used to heat the stomach and improve digestion; clove was believed to comfort the sinus; mace would prevent colic and bloody fluxes or diarrhea; nutmeg would benefit the spleen and relieve any bad cold.

Cinnamon, one of the most popular flavors in cooking, was considered to be particularly good for digestion and for sore throats. Hot pungent spices were used more liberally in winter diets or to treat cold diseases accompanied by excess phlegm. It is noteworthy that rheumatism was believed to be caused by abnormal rheum, or phlegm; the appropriate therapy would be pepper just as it is today, with the topical use of capsaicin - a chili pepper extract.

5. History of Spices

3000 BC to 200 BC

The Arabs develop the spice trade. For centuries, Arabs controlled this dangerous but lucrative trade, bringing pepper and cloves from India, cinnamon and nutmeg from the Spice Islands and ginger from China. They sold their spices in markets in Nineveh, Babylon, Carthage, Egypt and Rome. Caravans with as many as 4,000 camels traveled "the Golden Road of Samarkand" that stretched across the deserts of southern Asia and the Middle East between kingdoms in the East and markets in the West. The Bible refers to the spice trade in Genesis when Joseph's brothers sell him to a spice trading caravan bound for Egypt, and in the book of Kings when the Queen of Sheba pays tribute to King Solomon in spices, gold and precious stones. In 1453 BC the first Olympians are crowned with wreathes of laurel (bay leaves).

200 BC to 1200AD

The Romans dominate the trade. As their empire grew, Romans began sailing from Egypt to India to trade spices. It was a two-year voyage across the Indian Ocean to obtain pepper, cinnamon, nutmeg, cloves and ginger. During the reign of Tiberius Caesar, sailors discovered that the monsoon winds in the Indian Ocean blew east in the summer and west in the winter. By sailing with the trade winds, they could shorten their voyage to less than a year. There were constant losses from piracy and shipwreck, but the demand for spices made the trade highly profitable. Wealthy Romans valued spices as highly as gold. In 65 AD, they burned a year's worth of cinnamon in a funeral tribute to Nero's wife. When the Goths defeated Rome in 410, they demand a ransom of 3,000 pounds of pepper, along with gold, jewels and silk, to spare the life of the citizenry. Afterward, they required Rome to pay them 300 pounds of pepper per year.

1200 to 1500

As Europe began to develop in the Middle Ages, the demand for spices helped build world trade. Spices were so valued that they were used as currency. In the 11th century, pepper was counted out peppercorn by peppercorn and often used to pay taxes and rents. Europeans began to deal directly with Eastern merchants, and explorers were constantly seeking new trade routes. In 1271, a young Venetian named Marco Polo set out on a journey that would help open trade with the Orient and establish Venice as a major port. Venice remained dominant until around 1498 when the Spanish and Portuguese, weary of paying high prices, began their own explorations. Portuguese explorer Vasco De Gama sailed around Africa's Cape of Good Hope to reach Calcutta. He returned with pepper, cinnamon, ginger and a trading partnership with India. His voyages made tiny Portugal the richest nation in Europe. In 1492, Columbus discovered the Americas instead of a western route to the Spice Islands. He returned from the West Indies with allspice, vanilla and red peppers.

1400 to 1700

Demand for spices grew along with the middle classes during the Renaissance. The Portuguese remained dominant until the end of the 16th century when the Dutch made multiple expeditions to the East, establishing trade with local rulers. The Dutch conquered the city of Malacca in 1641, thus controlling the Malay Peninsula and nearby islands. In 1658 they gained control of the cinnamon trade in Ceylon. More Indonesian islands fell under their power, so that by the end of the17th century, they monopolized the Asian spice trade.

During the 1500s, the English sought to build their own spice trade. Elizabeth I chartered the British East India Company, which began to gain supremacy on the Indian mainland. In 1780, the Dutch and English fought a war, which was ruinous for the Dutch East India Company. By 1799, the Dutch had lost all spice trading centers and the Dutch East India Company closed. England became dominant; London's Mincing Lane was the spice-trading center of the world.

1600 to 1900

In 1672, Elihu Yale, a former clerk of the British East India Company in Madras, India, began his own spice business. He made a fortune and later founded Yale University. But America did not become a major force in the spice trade until 1798 when Captain Jonathan Carnes sailed into Salem, Massachusetts from Indonesia with a load of pepper. He had traded directly with the Indonesians rather than going through the European monopolies. Salem became the center of the American spice trade. More than a thousand ships made the trip to Sumatra during the next 90 years. Ultimately, pirates put America out of the spice trade. Ships were repeatedly robbed and the young government decided against backing its merchant ships in international waters. The U.S. relied on other countries for its supply.

6. SOURCES OF SPICES

Pepper, turmeric, cinnamon, cloves, mace and cardamom supplied from India. China and its neighboring countries supplied ginger, cassia, star anise, licorice and rhubarb. Chile peppers in addition to potatoes from Bolivia and Peru. The allure of trade for the valuable spices that could be transported successfully over vast distances was spurred by an increasing appetite in Europe for new spicy culinary experiences. The desire to monopolize major spices and the need to control the profitable sea routes were the driving forces that led to many of the dramatic events of history during the past 2000 years.

In ancient times, Arabia, Syria and Egypt provided well-organized marketing sites along the major recognized spice routes from which Asiatic spices were sent on their final land or sea journeys to the great spice ports of Europe, such as La Spezia, Venice and Genoa in Italy, Seville in Spain, Lisbon in Portugal, and the major port cities of England, Belgium and Holland.

Origins of Some Common Spices

India

cinnamon--Cinnamomum verum (= C. zeylanicum)--Lauraceae--bark; C. cassia inferior substitute
mace (aril), nutmeg (endosperm)--Myristica fragrans--Myristicaceae--nutmeg toxic in quantity
cloves--Syzygium aromaticum--Myrtaceae--flower buds--flavoring, antiseptic, tobacco; flavor can be synthesized
cardamom--Elettaria cardamomum--Zingiberaceae--pods--originally medicinal--Indian cooking, Danish pastry
pepper--Piper nigrum--Piperaceae--drupes, black or white depending on processing
turmeric--Cucurma longa--Zingiberaceae--root--yellow coloring

China

ginger--Zingiber officinale--Zingiberaceae--rhizome--powdered or candied
star anise--Illicium verum--Illiciaceae--immature fruit--as flavoring

Mediterranean, Mid- and Near East

bay leaf--Laurus nobilis--Lauraceae--leaves (do not eat them!)
caper--Capparis spinosa--Capparidaceae--flower buds--relish and flavoring
poppy--Papaver somniferum--probably from Medit.--flavoring, also narcotic
saffron--Crocus sativus--Iridaceae--stigmas--most costly condiment: 150,000 flowers/kg
sesame--Sesamum indicum (S. orientale)--Pedaliaceae--seeds, seed oil

New World

allspice--Pimenta dioica--Myrtaceae--fruits--flavor of cinnamon, cloves, nutmeg
red peppers, hot peppers, paprika--Capsicum annuum, etc.--Solanaceae--berries (capsaicin); C. frutescens--tabasco
vanilla--Vanilla planifolia--Orchidaceae--berry--only crop from large family; synthetic is inferior and often adulterated

The most important of the exotic spices in Medieval Europe was Indian pepper; this could be transported, stored and traded as peppercorns without any loss in its taste.

7. Culinary Herbs

While it is true that ancient recipes suggest that spices were added in extraordinary large amounts to banquet recipes, it is not clear how many people were meant to be served. It is likely that in practice large amounts were used only if a huge number of people were to participate in the feast. Thus, the actual amount of spice per individual may have been closer to what is acceptable today. Moreover, banquets were an opportunity to enjoy a prolonged bout of gorging, and it is likely that little food remained to be preserved from putrefaction over the ensuing post-banquet days. The evidence does not support claims that spice imports were driven by a need to either disguise the taste of spoiled food or to prevent putrefaction of cooked dishes. Furthermore, when coffee, tea, tobacco and snuff became fashionable in the 18th century, spices in food became less acceptable; thus, spice use declined in France and many other countries, even though methods for food preservation had not improved. It is noteworthy that honey was recognized to be an effective preserver of meats and other foods. In ancient times honey was applied to wounds, and more recent studies have shown it to be more effective than granulated sugar. Honey may have more than a simple osmotic effect that contributes to its bactericidal and fungicidal benefits. History records that when Alexander the Great died in Babylon, his body was encased in honey in a tomb for transfer to Alexandria for burial. There is no evidence that any spices are superior to or offer additive benefits to honey as a food preservative.

8. Spices as Aphrodisiac

Proprietary luxuries of this type, that consist of several dozen herbs and spices, are currently promoted as aphrodisiacs and tonics rather than as antidotes against poisoning, or as incenses, for appeasing the gods in religious ceremonies. Undoubtedly, spicy versions of these recipes that served the ancient pagan gods such as Priapus, Cupid, Venus, Eros, Pan and of course Aphrodite (the goddess who arose from sea foam - "aphros") continue to work their historic magic. Modern romances are catalyzed by spices and herbs which are called upon to provide symbolic and sensory support in luxurious perfumes, heady scents, and sensual aromatic cream or oil massages. However, it is of interest that the most appreciated of current aphrodisiacs is undoubtedly the New World's Aztec "food of the gods", the meso-American spice chocolate rather than the ancient and historic spices of Arabia and the Orient.

The essential oils and terpenoid alcohols of spices contribute to their smell, taste and tactile sensation. Thus, eugenol is found in cinnamon, clove and pimento; one of its medical qualities is a local anesthetic effect, which is utilized in dentistry. Menthol, from mints, has a cooling effect as well as a characteristic fresh taste and smell. Anise contains anethole, cinnamon produces cinnamaldehyde, mace contains myristin, and so on; all have specific pharmacological effects that are generally mild. However, some - such as myristicin - are more potent, and large doses can result in harmful effects such as hallucinations.

A number of spice chemicals are shared with herbs and flowers. It is noteworthy that colorful flowers result in an experience of exciting color and smell, whereas most spices result in excitatory sensations of taste and smell without being particularly stimulating to the visual sense. There are some exceptions, including the crocus which is the source of saffron, and edible flowers such as nasturtium which can spice up a salad. Similarly, chile peppers and radishes can be visually exciting, whereas cinnamon bark and cardamom seeds are relatively dowdy.

The following spices have had a long reputation of having aphrodisiacal properties.

Asafetida This has a foul smell, but in small amounts it can provide a sensual taste or smell. The same phenomenon applies to musk oil (from the musk ox) and castoreum (from the beaver), and perhaps to the secretions of the civet cat and the skunk: these agents can give a salty, animalistic, deeply erotic fragrant quality to a perfume when suitably diluted. Cardamom is popular in India and in Arabic cultures, and used to be employed by the Chinese court to give users a fragrant breath. Cloves and some other spices and herbs contain eugenol; its smell is fragrant and aromatic, and has long been considered as enhancing sexual feelings. Ginger contains gingerols, zingiberene and other characteristic agents that have made it a favored seductive flavor in Asiatic and Arabic herbal traditions. Mace and Nutmeg contain myristicin and similar compounds that are related to mescalin. In larger doses, nutmeg and mace can cause hallucinations, whereas in smaller amounts they are traditional aphrodisiacs. Pepper from India contains piperine: this pungent agent can stimulate sexual function, according to ancient beliefs. Saffron contains picrocrocin which is alleged to have the ability to cause erotic sensations. Vanilla contains the widely loved vanillin, whose taste and smell conjure up romantic feelings in the appropriate circumstances.

Other popular herbs that have been reported to have aphrodisiacal properties include garlic, mint, rosemary, sage and thyme. All these allegedly erotically stimulating agents have long been incorporated into cooking, incenses, rubs and other romantic sources for stimulation of sexual feeling. More recently, these and other herbs are utilized creatively in numerous massage oils and in incenses that are popularly utilized to improve sensations as a new-old form of therapy, with the modern title of aromatherapy.

9. PERFUMES AND INCENSES

From earliest history until today, fragrant, alluring smells have been regarded as essential elements of civilized relationships. Exotic plant odors and the scents that could be utilized for body application have inspired explorers, aristocrats, writers, poets, merchants and priests, and they have been of fundamental relevance to religious practices and to courtship. Many societies have felt that the burning of fragrant woods provides an ideal, ethereal token of appreciation to their gods. The liberation of incense smoke was a source of perfume: this word comes from the Latin per fumum, "by smoke". Incense is a word that means "that which is lit".

The sophisticated Greeks greatly appreciated such aromatic sources (aromata) as the turpentine tree, and this became an important import. They also valued the older Egyptian fragrant woods, and their exudates, such as those of myrrh, frankincense (olibanum) and cinnamon. Enormous amounts of money were spent on these exotic imports. The Greek island of Chios was the source of the valued gum exudate mastic as well as turpentine; the mastic was also used as a sort of chewing gum, and it gave rise to the word masticate. The more precious perfume incenses and spices came as imports through Arabia along well-established incense routes to be eagerly purchased by Mediterranean merchants who sold them to satisfy the increasing demands of markets throughout Europe. The most important ancient fragrances were frankincense and myrrh.

The Arabs used the milky sap of the frankincense tree, and called it al lubán, from the word for milk. (The same word gave rise to the name of Lebanon, whose mountains were always capped by milky snow). "Al lubán" became anglicized to olibanum, which is another name for frankincense; the latter name refers to the pre-eminence of this resin, the true or frank incense. Myrrh is a resin that has a bitter taste; its name is derived from Hebrew murr or maror, meaning bitter. Resins do not decay, and as shown by Majno, the resins of myrrh and similar agents are bacteriostatic. Myrrh continues to be used for this purpose in mouthwashes and toothpastes. Cinnamon, and the similar bark, cassia, when burned gives off a delightful fragrance; this is also readily obtained by grinding the bark. The phenolic compounds, such as cinnamic acid, are bacteriostatic, and fumes from their resins may well have served as fumigants as well as pleasing incenses.

10. Spices as Medicine

ANTIDOTES and MITHRIDATIUM

Poisoning was a favored means that was employed in ancient Greece and Rome to eliminate enemies. In the 1st century B.C., Mithridates VI, King of Pontus (located in present-day Turkey) worked with his physician to devise an effective antidote to all poisons. Two hundred years later, Galen wrote about antidotes, and he credited the King of Pontus with creating a 'mithridatium' that contained 41 ingredients. By that time other famous antidotes had been described; some of these persisted in use for centuries, including one devised by Galen. The most popular of the herbal antidotes besides mithridatium included galene, diascordium and philonium, which were named for their inventors. A generally used antidote that was alleged to be effective against venomous bites and stings was called theriaca; the theriacas of Damocrates and those produced in Cairo, Venice and other large cities became very popular.

The word 'theriaca' was corrupted to the word 'treacle' in English, especially for preparations of herbs in a thick, sweet base. The famous 17th century herbalist Nicholas Culpeper declared that the virtues and inexpense of garlic made it the 'poor-man’s treacle', and that it can be used as an effective panacea. Most of the other forms of theriacas and the various mithridatiums contained dozens of constituents, including exotic spices such as ginger, cinnamon, cassia, malabathrum, galbanum, cardamon, nard, pepper, frankincense, myrrh and saffron. Although these ineffective multiherb remedies remained in official use until the 19th century, they have spawned a host of similar tonics and stimulants that contain a comparable, illogical array of herbs and spices that enjoy a wide market today. See section on Spices as Aphrodisiacs.

The only differences in today’s theriaca equivalents are the incorporation of various modern constituents such as vitamins, minerals, amino-acids and newly fashionable herbs. A similar group of medical recipes included bitters or 'hiera', which were introduced in Greece for use in the Temples of Ascalepios. The components and the number of constituents varied considerably over the ages, although aloes and cinnamon were commonly used. These were prescribed as purgatives and tonics, and were eventually recommended as valued panaceas for a great number of different disorders. Their use persisted 'despite no evidence of effectiveness ' for many centuries. Today, some European countries still make available similar bitter tonics (such as the ancient Hiera picra or holy bitter), and they are marketed as non-specific remedies; people regard them as digestives, cough medicines and so on.

11.Anti-microbial Functions of Spices

In all medical systems of Asia and Europe, spices have been used both as therapeutic foods and as medicines. Despite the contrasting opinions of different experts who insisted on their indications, there is little evidence of any specific benefit from most spices. Many pungent spices are unattractive to animals (excepting most, humans, many birds and some rodents), and they do have some antimicrobial, gastrointestinal, and mucus-loosening properties.

Billing J, Sherman PW. an evolutionary biologist and professor of neurobiology and behavior at Cornell, in his article (Rev Biol. 1998 Mar;73(1):3-49), "Antimicrobial functions of spices: why some like it hot" describes a study on this subject. The study addressed the facts - the varied approach in food preparation throughout the world, patterns of spice usage among various cultures and countries - What factors underlie these differences? Why are spices used at all? To investigate these questions and to establish the bacteria-spices connection, a study was conducted.

Sherman credits Billing, a Cornell undergraduate student of biology at the time of the research, with compiling many of the data required to make the bacteria-spices connection: A total of 4,578 recipes from 93 cookbooks representing the frequency of use of 43 spices in traditional cuisines of 36 countries; the temperature and precipitation levels of each country; the horticultural ranges of 43 spice plants; and the antibacterial properties of each spice.

These data were used to investigate the hypothesis that spices inhibit or kill food-spoilage microorganisms. In support of this is the fact that spice plant secondary compounds are powerful antimicrobial (i.e., antibacterial and antifungal) agents.

"The proximate reason for spice use obviously is to enhance food palatability," says Sherman, . "But why do spices taste good? Traits that are beneficial are transmitted both culturally and genetically, and that includes taste receptors in our mouths and our taste for certain flavors. People who enjoyed food with antibacterial spices probably were healthier, especially in hot climates. They lived longer and taught their offspring and others. We believe the ultimate reason for using spices is to kill food-borne bacteria and fungi."

In general it is claimed, Garlic, onion, allspice and oregano were found to be the best all-around bacteria killers - the most potent antibacterial and antifungal agents;(they kill everything), followed by thyme, cinnamon, tarragon and cumin (any of which kill up to 80 percent of bacteria). Capsicums, including chilies and other hot peppers, are in the middle of the antimicrobial pack (killing or inhibiting up to 75 percent of bacteria), while pepper of the white or black variety inhibits 25 percent of bacteria, as do ginger, anise seed, celery seed and the juices of lemons and limes.

However, there is lack of uniformity in findings, and this may reflect non-uniformity in source material. Furthermore, some fungi and bacteria use spices as supportive media for their growth. Although it is often claimed that exotic spices were sought as valuable food preservatives, this is not correct. Thus, simple pickling with common-place vinegar, garlic and mustard can preserve and flavor food almost as well as dehydrating and salting can. Honey and strong sugar soultions can also be used as food preservatives.

There is little evidence that pepper, cloves, nutmegs, ginger and other expensive spices were used as alternatives to garlic, etc. to preserve food or to delay the spoilage of cooked dishes. Their use in their countries of origin is not related to spices serving as an alternative to refrigeration, since they are usually added to fresh foods as flavors. In particular, they add zest to a bland diet based on rice and other high-carbohydrate vegetable staples. Indeed, the concentrations of spices that would be needed to significantly retard food spoilage by microorganisms would result in an overwhelming flavor, that may be worse than that of the decaying food.

However the micronutrient hypothesis - that spices provide trace amounts of anti-oxidants or other chemicals to aid digestion - could be true and still not exclude the antimicrobial explanation, Sherman says. However, this hypothesis does not explain why people in hot climates need more micro-nutrients, he adds. The antimicrobial hypothesis does explain this.

Top 30 Spices with Antimicrobial Properties:

* 1. Garlic
* 2. Onion
* 3. Allspice
* 4. Oregano
* 5. Thyme
* 6. Cinnamon
* 7. Tarragon
* 8. Cumin
* 9. Cloves
* 10. Lemon grass
* 11. Bay leaf
* 12. Capsicums
* 13. Rosemary
* 14. Marjoram
* 15. Mustard

* 6. Caraway
* 17. Mint
* 18. Sage
* 19. Fennel
* 20. Coriander
* 21. Dill
* 22. Nutmeg
* 23. Basil
* 24. Parsley
* 25. Cardamom
* 26. Pepper (white/black)
* 27. Ginger
* 28. Anise seed
* 29. Celery seed
* 30. Lemon/lime

12. Business of spices

Within the past one decade the international trade in spices has grown by leaps and bounds. An estimated 500,000 tonnes of spices and herbs valued at 1500 million US dollars are now imported globally every year. An impressive 46% of this supply comes from India. India's exports of spice extracts have shown spectacular growth attaining over 50 percent of the global market within a short span. Over the past decade, the Indian Spices industry has made quality the cutting edge of its global game plan. In recent years, export of Indian Spices has been taking giant leaps. The Indian export of spices has crossed the 450 million US dollar mark during 1999-2000 and has reached 468 million US dollar. This remarkable achievement is born of a sea change in the industry scenario. From traditional commodity exports, Indian Spices have evolved into a state-of-the-art industry. Absorbing technology, broad basing its products range, developing value added products, identifying niche markets, forging strategic alliances clinching global collaborations and joint ventures.

13. The Indian share

At present, India produces around 2.5 million tonnes of different spices valued at approximately 3 billion US $, and holds the premier position in the world. Because of the varying climates suitable for the spice cultivation. Almost all spices are grown in this country. In almost all of the 25 states and seven union territories of India, at least one spice is grown in abundance. No country in the world produces as many kinds of spices as India.

In recent years, export of Indian Spices has been taking giant leaps. The Indian export of spices has crossed the 450 million US dollar mark during 1999-2000 and has reached 468 million US dollar. This remarkable achievement is born of a sea change in the industry scenario. From traditional commodity exports, Indian Spices have evolved into a state-of-the-art industry. Absorbing technology, broad basing its products range, developing value added products, identifying niche markets, forging strategic alliances clinching global collaborations and joint ventures.

State

Product

ANDHRA PRADESH
Chilly,Ginger,Mustard,Turmeric

ARUNACHAL PRADESH
Ginger ,Tejpat, Turmeric

ASSAM
Aniseed, Turmeric

HARYANA
Garlic

BIHAR
Ajovan,Garlic, Mustard,Turmeric

GUJARAT
Chilly,Cumin,Dill Seed,Fennel,Fenugreek,Garlic

HIMACHAL PRADESH
Ginger

JAMMU & KASHMIR
Ajovan,Saffron

KARNATAKA
Cardamom (Small),Chilly,Clove,Garlic,Ginger ,Kokam,Nutmeg & Mace,Pepper,Turmeric,Vanilla

KERALA
Cardamom (Small),Cinnamon & Cassia,Clove,Ginger ,Nutmeg & Mace,Pepper,Turmeric,Vanilla

MADHYA PRADESH
Chilly,Garlic,Ginger

MAHARASHTRA
Chilly,Garlic,Pomegranate Seed,Turmeric

MEGHALAYA
Ginger ,Turmeric

MIZORAM
Ginger

ORISSA
Chilly,Garlic,Ginger ,Turmeric

PUNJAB
Aniseed,Celery

RAJASTHAN
Chilly,Cumin,Coriander,Dill Seed,Fennel,Fenugreek,Garlic

SIKKIM
Cardamom (Large),Ginger ,Tejpat

TAMIL NADU
Cardamom (Small),Chilly,Cinnamon & Cassia,Clove,Ginger ,Herbal & Exotic Spices,Nutmeg & Mace,Pepper,Pomegranate Seed,Turmeric,Vanilla

TRIPURA
Turmeric

UTTAR PRADESH
Aniseed,Celery,Chilly,Coriander,Cumin,Fennel,
Fenugreek,Garlic, Mustard,Turmeric

WEST BENGAL
Cardamon (Large),Chilly,Ginger ,Turmeric

In April 1998, ‘Food Ingredients Asia’, exhibition was held in Shanghai, China. This paved the way for natural spices from India to emerge in Chinese Market. Spices Board India’s presence helped the Chinese see the many kinds of Indian Spices.

The Spices Board India (Ministry of Commerce, Government of India) is the apex body for the export promotion of Indian Spices. Established in 1987, the Board is the catalyst of these dramatic transitions. The Board has been with the Indian Spice industry every step of the way. The Board plays a far reaching and influential role as a developmental, regulatory and promotional agency for Indian Spices.

Indias's share in world trade of spices
(2002 - 2003)

SCIENTISTS SPEAK ABOUT DNA

2005-06-21T21:45:00.000-07:00

SCIENTISTS SPEAK ABOUT DNA

"[The instructions within the DNA of the cell] if written out, would fill a thousand 600-page books. Each cell is a world brimming with as many as two hundred trillion tiny groups of atoms called molecules . . Our 46 chromosome `threads' [in one DNA molecule] linked together would measure more than six feet. Yet the nucleus that contains them is less than four ten-thousandths of an inch in diameter."—*Rick Gore, "The Awesome Worlds Within a Cell," National Geographic, September 1976, pp. 357-358, 360.

Introduction to DNA Extractions

2005-06-01T19:52:00.000-07:00

Introduction to DNA Extractions

Introduction to DNA Extractions
Lana Hays
Access Excellence Fellow
Simon Kenton H. S.
11132 Madison Pike
Independence, KY 41051
AELHays@aol.com

Notes to the teacher:
Through two weeks of numerous trials, I have learned a great deal about DNA extractions. I originally worked with eight different protocols to modify, simplify, and develop good extraction labs. My intent was to produce simple DNA extractions that use various types of cells. The materials used come from the grocery store, health food stores, and butcher shops. Several extractions require a centrifuge. I made it a point to get a centrifuge this year so that I could run the experiments. If you do not have access to a centrifuge, you might run the extractions as written and try to create ways to get around it. The centrifuge step can be skipped in the thymus experiment and good results still obtained. It's amazing what steps can be eliminated or modified.

It is best to begin collecting the materials two weeks in advance. All materials that you extract from must as fresh as possible. The two most difficult items to obtain are non-roasted wheat germ and calf thymus. Health food stores usually carry the non-roasted type of wheat germ, as do some large grocery stores. Thymus (sweetbread) will need to be ordered from a butcher shop. As butcher shops don't always know what will be slaughtered ahead of time, I had several shops trying to get it. Liver is not difficult to get but should be ordered fresh. Once purchased, thymus can be frozen until you need them. Cut them into chunks before freezing so that you can get just what you need each day. The other items can be purchased at most any large grocery store.

I like for my students to do as much of the protocol as they can. With several blenders and a little organization, all the labs can be completed in a 55 minute period or less. Each protocol produces enough lysate for 10-15 spoolings. If you are short on time and equipment, you can demonstrate the first part and then have the students complete the rest of the lab. If you want the individual students to complete all of the protocol, cut the quantities down proportionatey until you get to the spooling step. At this point, use the original protocol. The blender can handle mixing 10-15 ml of solution but you may need to cut the blending time down.

When DNA extractions are performed, you can expect three basic results.

1. No DNA
2. DNA appears fluffy which means it has sheared in the extraction process
3. DNA appears as thin threads.

Although DNA that strands is the most impressive, DNA that has sheared still shows that DNA is present.

All the experiments will yield DNA but some more than others. The lima bean bacteria, and yeast give the poorest results. I'm sure that with some more experimentation they could be improved greatly. The most impressive is the calf thymus, so I have students do it last. The long threads of DNA are easily spooled and the quantity is immense compared to the other extractions.

If you have students do many DNA extractions, you will find that their lab skills will improve. However, a problem that constantly persists occurs when they add the alcohol--the students usually pour it too fast. Rather than forming two distinct layers, they mix the two. Once that happens, there's not much that can be done.

The experiments work well as written. However the following substitutions can be used:

1. a reusable coffee filter or cheese cloth can be used to strain the materials

2. 91-99% isopropyl (rubbing alcohol) can be substituted for ethanol,although I prefer ethanol

3. fresh papaya or pineapple juice can be substituted for the meat tenderizer solution (use the same amount of ml as the meat tenderizer solution)

4. 10% SDS (sodium dodecyl sulfate) can be used in place of all the detergent solutions. It comes as a 10% solution already mixed or you can buy the powder and mix a 10% solution (5g SDS and 50ml distilled water).

A good science project for students is to run through several of the protocols. Then have them design modifications to test. They can use the substitutions, use other things to extract from, or switch solutions/protocols. You can have the students pipet the alcohol/DNA layer off and place it in a clean test tube to view later. At the end, they can compare the DNA from all the extractions or even create other labs in which to use the DNA. An excellent experiment is to have students run all the DNA extractions except yeast. Have them analyze the other extractions using the summary chart and research the characteristics of yeast. Based on their findings, have them design a protocol for yeast DNA extraction, run the experiment, and justify their results.

There are two to three basic steps in DNA extraction. The cell must be lysed (broken open) to release the nucleus. The nucleus (if present) must also be opened to release the DNA. At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Once the DNA is released, it must then be precipitated in alcohol.

In order for the cell to be lysed, the lipid walls must be broken down. The detergent and salt solutions accomplish this. Cell walls, cell membranes, and nuclear membranes are also broken down by the action of the blender. In all but one protocol I eliminated the use of heat. Some references state that a temperature of 60oC is necessary to denature the DNAase enzymes that cause shearing in DNA while DNA is denatured about 80oC. Other references state that DNA can denature at 60oC. From all the experiments I ran (except for the wheat germ protocol), I had sheared DNA when I used heat. Heat may destroy the enzymes as well as the DNA. However keeping the solutions cool seems to slow the enzyme action. The prep solution uses epsom salts and buffered aspirin to further deactivate the enzymes that degrade DNA when released and stabilize the DNA (acid vs. base). Sodium bicarbonate (baking soda) also is used to buffer the solution. The meat tenderizer has papain, an enzyme that helps clean the protein from the DNA that can contaminate it. Papaya juice and pineapple juice also contains this enzyme. Finally, the ethanol is used to precipitate the DNA. In water, DNA is soluble. When it is in ethanol, it uncoils and precipitates leaving behind the other cell components that are not soluble in ethanol.

All in all, the DNA extraction labs are very workable. Try some and then decide if you would like to modify any to fit your needs better. Good luck!!

* Onion DNA Extraction
* Wheat Germ DNA Extraction
* Lima Bean Bacteria DNA Extraction
* Yeast DNA Extraction
* Thymus DNA Extractions
* DNA Extraction Summary Chart
* Experimental Design: Yeast Extraction
* Credits and References

Onion DNA Extraction
Materials

* fresh onions
* graduated cylinders (10ml and 100ml)
* knife
* 15 ml test tube
* blender
* test tube rack or 250 ml beaker
* strainer
* glass stirring rod
* coffee filters
* non-iodized salt
* Adolph's natural meat tenderizer
* Palmolive detergent
* beaker
* distilled water
* ice cold 95% ethanol

Solutions
Detergent/salt solution:

* 20 ml detergent
* 20 g non-iodized salt
* 180 ml distilled water

5% meat tenderizer solution:

* 5 g meat tenderizer
* 95 ml distilled water

Protocol

1. Cut an inch square out of the center of 3 medium onions. Chop and place in a blender.

2. Add 100 ml of detergent/salt solution.

3. Blend on high 30 sec-1 minute.

4. Strain the mixture into a beaker using a strainer with a coffee filter.

5. Add 20-30 ml meat tenderizer and stir to mix.

6. Place 6 ml filtrate in a test tube.

7. Pour 6 ml ice cold ethanol carefully down the side of the tube to form a layer.

8. Let the mixture sit undisturbed 2-3 minutes until bubbling stops.

9. The DNA will float in the alcohol. Swirl a glass stirring rod at the interface of the two layers to see the small threads of DNA.

Modified from: "Isolation of DNA from Onion" Ellen Averill

Wheat Germ DNA Extraction
Materials

* 250 ml beaker
* baking soda
* hot plate
* Adolph's natural meat tenderizer
* non-roasted wheat germ
* ice cold 95% ethanol
* thermometer
* 15 ml test tube
* pH meter
* glass stirring rod
* Palmolive
* detergent
* distilled water
* test tube rack or 250 ml beaker
* graduated cylinders (10ml and 100ml)

Solutions
Baking soda solution:

* Add baking soda to distilled water until a pH of approximately 8.0 is reached.

Protocol

1. Add 100 ml distilled water to a beaker and heat to 50-60oC.

2. Add 1.5 g wheat germ and mix until dissolved.

3. Add 5 ml detergent. Maintain 50-60oC temperature and stir for 5 minutes.

4. Add 3 g meat tenderizer.

5. Add baking soda solution to bring the pH to approximately 8.0.

6. Maintain the 50-60oC temperature and stir for 10 minutes.

7. Remove from heat.

8. Add 6 ml of the solution to a test tube and cool to room temperature.

9. Pour 6 ml ice cold ethanol carefully down the side of the tube to form a layer.

10. Let the mixture sit undisturbed 2-3 minutes until bubbling stops.

11. The DNA will float in the alcohol. Swirl a glass stirring rod at the interface of the two layers to see the small threads of DNA.

Modified from: "Wheat Germ DNA Extraction" Judy Brown

Lima Bean Bacteria DNA Extraction
Materials

* dry lima beans
* Palmolive detergent
* centrifuge
* distilled water
* centrifuge tube
* fresh papaya juice
* graduated cylinder (10ml)
* non-iodized salt
* granulated sugar
* pipet
* epsom salts
* 15 ml test tube
* bufferin (325mg)
* test tube rack or 250 ml beaker
* ice cold 95% ethanol
* glass stirring rod

Solutions
Lima Bean Bacteria Suspension: Place 1-2 handfuls of dry lima beans in a large jar and fill halfway to the top with distilled water. Cover and sit in a warm room for 2-3 days. Culturing longer than three days often results in more DNA but it usually shears. Pour through a strainer and keep the liquid for the extractions.

Prep buffer solution:

* 57 g granulated sugar
* 3 g epsom salts
* 1 buffered aspirin
* add distilled water for a total volume of 500 ml

50% detergent solution:

* 20 ml detergent
* 20 ml distilled water

Salt solution:

* 29.2 g non-iodized salt
* add distilled water for a total volume of 250 ml

Protocol

1. Add 14 ml of the bacterial suspension to a centrifuge tube and spin in a balanced centrifuge for 5 minutes.

2. Pour off the liquid (supernatant) and discard. You want to keep the pellet as this has your cells.

3. Add 5 ml of prep buffer and resuspend your cells with a pipet.

4. Add 1 ml 50% detergent solution.

5. Add 1 ml papaya juice.

6. Add 2 ml salt solution and shake for 2 minutes.

7. Place the tube in the centrifuge and spin for 5 minutes. Make sure the centrifuge is balanced.

8. Draw off 7 ml of the supernatant (liquid) as this has the DNA and place it in a clean test tube.

9. Pour 7 ml of ice cold ethanol carefully down the side of the tube.

10. Let the mixture sit undisturbed 2-3 minutes until the bubbling stops.

11. The DNA will float in the alcohol. Swirl a glass rod at the interface of the two layers. You may see some tiny threads of DNA but are more likely to see fluffy, white sheared DNA.

Modified from: "Generic, All Purpose DNA Extraction from Meat Protocol" Judy Brown

"Mammalian DNA Extraction" Theresa Knapp

Yeast DNA Extraction
Materials

* dry yeast
* Adolph's natural meat tenderizer
* beaker
* distilled water
* non-iodized salt
* glass stirring rod
* Palmolive detergent
* graduated cylinders (10ml and 100ml)
* blender
* 15 ml test tube
* ice cold 95% ethanol
* test tube rack or 250 ml beaker

Solutions
detergent/salt solution:

* 20 ml detergent
* 20 g non-iodized salt
* 180 ml distilled water

5% meat tenderizer solution:

* 5 g tenderizer
* 95 ml distilled water

Protocol

1. Mix 1 package of dry yeast with 40 ml of 50oC hot tap water to dissolve the yeast in a beaker. Keep mixture covered and warm for about 20 minutes.

2. Add 40 ml detergent/salt solution.

3. Place mixture in a blender and blend 30 sec-1 minute on high.

4. Pour mixture back into the beaker, add 15 ml of meat tenderizer solution, and stir to mix.

5. Place 6 ml of mixture into a test tube.

6. Pour 6 ml of ice cold ethanol carefully down the side of the tube to form a layer.

7. Let the mixture sit undisturbed 2-3 minutes until bubbling stops.

8. You will see a precipitate in the alcohol. Swirl a glass stirring rod at the interface of the two layers. The precipitate is DNA.

Modified from: "Isolation of DNA from Onion" Ellen Averill

Thymus DNA Extractions
Materials

* fresh thymus
* blender
* beaker
* sugar
* pipet
* centrifuge tube with cap
* bufferin (325mg)
* knife
* graduated cylinders (10ml,100ml)
* epsom salts
* distilled water
* centrifuge
* 95% ice cold ethanol
* 15 ml test tubetest tube rack or beaker
* Palmolive detergent
* non-iodized salt

Solutions
prep buffer solution:

* 57 g granulated sugar
* 1 buffered aspirin
* 3 g epsom salts
* add distilled water for a total of 500 ml

10% detergent solution:

* 90 ml distilled water
* 10 ml Palmolive detergent

salt solution:

* 29.2 g non-iodized salt
* add distilled water for a total volume of 250 ml

Protocol

1. Cut out a chunk of liver or thymus 1 inch square and place in the blender.

2. Add 100 -150 ml prep buffer and 10 ml detergent solution to the blender.

3. Blend for 1 minute or until the mixture is smooth.

4. Pour the mixture into a beaker.

5. Transfer 1 ml of the mixture to a centrifuge tube.

6. Add 2 ml of salt solution, cap, and shake for 2 minutes.

7. Centrifuge for 7 minutes in a balanced centrifuge.

8. Carefully remove the tube from the centrifuge and note the two layers:
* lower layer - pellet
* *upper layer - liquid (supernatant) and what has the DNA in it.

9. Pipet or carefully pour the liquid into a clean test tube.

10. Pour 5 ml ice cold ethanol carefully down the side of the tube to form a layer.

11. Let the mixture sit undisturbed for a minute or two.

12. The DNA will float in the alcohol. The DNA of the thymus will be long threads that easily spool.

Modified from: "Generic, All Purpose DNA Extraction from Meat Protocol" Judy Brown

"Mammalian DNA Extraction" Theresa Knapp

DNA Extraction Summary Chart
QUESTIONS ONION WHEAT GERM BACTERIA YEAST THYMUS
What are the cell characteristics?

What lyses the cell and nucleus?

What protects the DNA?

What precipitates the DNA?

Amount of DNA

Description of DNA

Changes in protocol

Brief procedure
Experimental Design: Yeast Extraction
Task:
You are to design an experiment to extract DNA from yeast, run the experiment, and then do a lab write-up with your results.

Procedure:

1. Investigate what type of cells yeast are as well as their cell characteristics.
2. Study the protocols for onion, wheat germ, bacteria, and thymus.
3. Based on what you have learned about yeast and its characteristics, design an experiment to extract DNA from your yeast.
4. Run the experiment.
5. Complete a lab write-up that includes:

A. Protocol
B. Justification of protocol used
C. Results and conclusions

Credits and References

* "Isolation of DNA from Onion"
Ellen Averill
Kendrick High School
6015 Georgetown Drive
Columbus, GA 31907
*Published in Woodrow Wilson's Biology Module 1993 A Further Look at Biotechnology

* "Wheat Germ DNA Extraction"
"Onion DNA Extraction"
"Generic All Purpose DNA Extraction from Plants Protocol"
"Generic All Purpose DNA Extraction from Animal Protocol"
"DNA Yeast Extraction"
Judy Brown
Edison Career Center
Montgomery County, Maryland

* "Mammalian DNA Extraction"
Theresa Knapp
Stevenson High School
Lincolnshire, IL 60069

* "E. coli DNA Extraction"
Rod Best
Sparta, North Carolina
*Published in NABT Biotechnology Sourcebook

* "The Cookbook Translator"
Lana Hays
Simon Kenton High School
Independence, KY 41051
and
Anthony Bertino
Canandaigua Academy
Canandaigua, NY 14424
*Published in Woodrow Wilson Biology Module 1993
A Further Look at Biotechnology

How Does It Work?

2005-06-01T19:29:00.000-07:00

How Does It Work?

How Does It Work?
Wheat germ

This is the DNA source in this protocol. Wheat germ comes from wheat seeds. The "germ" is the embryo, which is the part of the seed that can grow into a new wheat plant. When wheat seeds are milled into white flour, the wheat germ and wheat bran are removed, leaving only starch. Wheat germ contains many nutrients while wheat bran consists of fiber. Whole wheat flour contains all parts of the wheat seed and is therefore more nutritious than white flour while also providing important fiber for digestion.
cross section of a wheat seed
Water temperature

The heat softens the phospholipids (fats) in the membranes that surround the cell and the nucleus. It also inactivates (denatures) the deoxyribonuclease enzymes (DNase) which, if present, would cut the DNA into such small fragments that it would not be visible. Denatured enzymes and DNA unravel, lose their shape, and thus become inactive. Enzymes denature at 60° Celsius, and DNA denatures at 80° Celsius.
Detergent

Detergent contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It acts the same way in the DNA extraction protocol, pulling apart the fats (lipids) and proteins that make up the membranes surrounding the cell and nucleus. Once these membranes are broken apart, the DNA is released from the cell.

Soap molecules and grease molecules are made of two parts:

* Heads, which like water
* Tails, which hate water.

Both soap and grease molecules organize themselves in bubbles (spheres) with heads outside to face the water and tails inside to hide from the water.
grease and soap

When soap comes close to grease, it captures it, forming a greasy soapy ball.

A cell's membranes have two layers of lipid (fat) molecules with proteins going through them.
cell membrane

When detergent comes close to the cell, it captures the lipids and proteins and releases the DNA.
cell + detergent
Alcohol

The DNA released from the cell nucleus is dissolved in the water/detergent/wheat germ solution and cannot be seen. DNA precipitates out of solution in alcohol, where it can be seen. Besides allowing us to see the DNA, the alcohol separates the DNA from the other cell components, which are left behind in the water solution.

How to Extract DNA from Anything Living

2005-06-01T19:26:00.000-07:00

How to Extract DNA from Anything Living

How to Extract DNA from Anything Living

DNA! You mean I can see it? How?

Just follow these 3 easy steps:
Detergent
eNzymes (meat tenderizer)
Alcohol

It's that simple? Tell me more!

First, you need to find something that contains DNA. Since DNA is the blueprint for life, everything living contains DNA.
green split peas

For this experiment, we like to use green split peas.

But there are lots of other DNA sources too, such as:

* Spinach
* Chicken liver
* Onions
* Broccoli

Here's the fun part. Put in a blender:

* Your DNA source (about 100ml or 1/2 cup of split peas)
* A large pinch of table salt (less than 1ml or 1/8 teaspoon)
* Twice as much cold water as the DNA source (about 200ml or 1 cup)

blending DNA

Blend on high for 15 seconds.

The blender separates the pea cells from each other, so you now have a really thin pea-cell soup. Because this step is pretty messy, certain sources of DNA should not be used, such as:

* Your family pet, Fido the dog
* Your little sister's big toe
* Bugs you caught in the yard

And now, those 3 easy steps:
straining

1. Pour your thin pea-cell soup through a strainer into another container (like a measuring cup).

How much pea soup do you have? Add about 1/6 of that amount of liquid detergent (about 30ml or 2 tablespoons) and swirl to mix. Let the mixture sit for 5-10 minutes.

Pour the mixture into test tubes or other small glass containers, each about 1/3 full.

liquid detergents

Try one of these detergents or whatever you have on hand.

Why am I adding detergent?

meat tenderizer

2. Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see.

Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens cleaning solution.

What is an enzyme?

pouring alcohol

3. Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.

DNA will rise into the alcohol layer from the pea layer. You can use a wooden stick or other hook to draw the DNA into the alcohol.

What is that stringy stuff?

Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide:
"Hmmm...which layer should I go to?"

This is sort of like looking around the room for the most comfortable seat. Some will choose the couch, others might choose the rocking chair.

In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer.

DNA is a long, stringy molecule that likes to clump together.

Congratulations! You have just completed a DNA extraction!

Now that you've successfully extracted DNA from one source, you're ready to experiment further. Try these ideas or some of your own:

* Experiment with other DNA sources. Which source gives you the most DNA? How can you compare them?
* Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?
* Experiment with leaving out or changing steps. We've told you that you need each step, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.
* Do only living organisms contain DNA? Try extracting DNA from things that you think might not have DNA.

DNA Extraction from Wheat Germ

2005-06-01T19:23:00.000-07:00

DNA Extraction from Wheat Germ

DNA Extraction from Wheat Germ

Would you like to see LOTS of DNA?

This method yields large quantities of DNA that can easily be collected.

Here's what you'll need for each DNA extraction:
Materials Needed:

Raw wheat germ - 1 gram or 1 teaspoon.
Wheat germ can be purchased at a health food store or some large supermarkets; toasted wheat germ does not work.

Liquid detergent - 1 ml or a scant 1/4 teaspoon
The following liquid soap products have been tested and found to work well for this DNA extraction protocol: Lemon Fresh Joy, Woolite, Ivory, Shaper, Arm & Hammer, Herbal Essence shower gel by Clairol, Tide, Dish Drops, Kool Wash, Cheer, Sunlight Dish Soap, Dawn, Delicate, All, and Ultra Dawn.

The following liquid products do not work well: Life Tree, Shout, Shaklee, Sunlight Dishwasher, and LOC. Powdered detergents also do not produce good results with this protocol.

Alcohol - 14 ml or 1 tablespoon
70% isopropyl alcohol ("Rubbing alcohol") is the least expensive since it can be purchased at a grocery store or pharmacy. However, it contains a higher percentage of water, making it slightly more difficult to precipitate the DNA.

95% ethyl alcohol and Everclear grain alcohol (which is 95% alcohol) both work equally well. The DNA is easy to collect.

50-60° Celsius tap water - 20 ml or 1 tablespoon
Do not use water hotter than 50-60° C. The water will cool during the extraction procedure, but this does not matter. Test your tap water -- it may be hot enough right from the tap.

50 ml test tube
Capped test tube, beaker or spice jar.

Graduated cylinder,
Measuring spoons, or other measuring devices.

Wooden applicator stick,
Glass stirring rod/hook, paper clip hook, or shish kebob skewer for stirring and collecting the mixture.

Eyedropper,
Pasteur pipette and bulb, or pieces of paper towel - may be needed to remove foam

Sealable container (optional),
Such as a tube, vial or jar to store DNA.

50% alcohol (optional) - for storing DNA.
You can use either isopropyl alcohol (rubbing alcohol) or ethyl alcohol for storing the DNA you extract. To make 100 ml of 50% alcohol with isopropyl alcohol, mix 71 ml of 70% isopropyl alcohol (rubbing alcohol) with 29 ml of distilled water. Using ethyl alcohol or Everclear grain alcohol, mix 53 ml of 95% ethyl alcohol (ethanol) with 47 ml of distilled water.

Paper towels
or filter paper - for drying DNA
Instructions

1.

Place 1 gram or 1 teaspoon of raw wheat germ in a 50 ml test tube, beaker or jar.
2.

Add 20 ml or 1 tablespoon of hot (50-60 °C) tap water and mix constantly for 3 minutes.
3.

Add 1 ml or a scant 1/4 teaspoon of detergent and mix gently every minute for 5 minutes. Try not to create foam.
4.

Use an eyedropper, pipette, or piece of paper towel to remove any foam from the top of the solution.
5.

Tilt the test tube, beaker or jar at an angle. SLOWLY pour 14 ml or 1 tablespoon of alcohol down the side so that it forms a layer on top of the water/wheat germ/detergent solution. Do not mix the two layers together. DNA precipitates at the water-alcohol interface (the boundary between the water and the alcohol). Therefore, it is crucial to pour the alcohol very slowly so that it forms a layer on top of the water solution. If the alcohol mixes with the water, it will become too dilute and the DNA will not precipitate.
6.

Let the test tube, beaker or jar sit for a few minutes. White, stringy, filmy DNA will begin to appear where the water and alcohol meet. You will usually see DNA precipitating from the solution at the water-alcohol interface as soon as you pour in the alcohol. If you let the preparation sit for 15 minutes or so, the DNA will float to the top of the alcohol.

You can usually get more DNA to precipitate from the solution by using one of the DNA-collecting tools (such as a glass or paper clip hook) to gently lift the water solution up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate. You may find it helpful to pour the water/detergent solution into a clean test tube, leaving behind the wheat germ, before adding the alcohol.
7.

Use a glass or paper clip hook or a wooden stick to collect the DNA.
8.

If you want to keep the DNA, store it in 50 - 70% alcohol in a sealed tube or air dry it on paper towels or filter paper.

Journal of Biological Chemistry

2005-05-09T20:40:00.000-07:00

Journal of Biological Chemistry

Human Traits

2005-04-17T23:48:00.000-07:00

Human Traits

Human Traits: autosomal

1. Shape of face (probably polygenic)

Oval dominant, square recessive

2. Cleft in chin

No cleft dominant, cleft recessive

3. Hair curl (probably polygenic)
Assume incomplete dominance

Curly: homozygous
Wavy: heterozygous
Straight: homozygous

4. Hairline

Widow peak dominant, straight hairline recessive

5. Eyebrow size

Broad dominant, slender recessive

6. Eyebrow shape

Separated dominant, joined recessive

7. Eyelash length

Long dominant, short recessive

8. Dimples

Dimples dominant, no dimples recessive

9. Earlobes

Free lobe dominant, attached recessive

10. Eye shape

Almond dominant, round recessive

11. Freckles

Freckles dominant, no freckles recessive

12. Tongue rolling

Roller dominant, nonroller recessive

13. Tongue folding

Inability dominant, ability recessive

14. Finger mid-digital hair

Hair dominant, no hair recessive

15. Hitch-hiker's thumb

Straight thumb dominant, hitch-hiker thumb
recessive

16. Bent little finger

Bent dominant, straight recessive

17. Interlaced fingers

Left thumb over right dominant, right over left recessive

18. Hair on back of hand

Hair dominant, no hair recessive

19. Tendons of Palmar Muscle

Two tendons dominant, three tendons recessive

Human Disease Genes That Are Found in Flies, Worms, & Yeast

2005-04-12T06:20:00.000-07:00

Human Disease Genes That Are Found in Flies, Worms, & Yeast

Unity of Life
Human Disease Genes That Are
Found in Flies, Worms, & Yeast

Flies don't get kidney disease, and worms don't get heart disease, yet many of the human genes that are faulty in these and other human disorders have parallel genes in model organisms, where they can be studied more easily.

After the fly's genome was sequenced in March 2000, a team of scientists found that 61 percent of the human genes known to be mutated in 289 human diseases have close equivalents in flies. Many of these genes also have parallels in worms and even in yeast.

The extent of similarity between some of the human disease genes and genes that have been sequenced in flies, worms, and yeast is shown below. Genes with the highest degree of sequence similarity are indicated by the filled circle. The next group is two-thirds. And a lower degree of similarity is shown by the one-third filled circle. Empty circles indicate extremely low or no similarity between the genes.

"Some of the human disease genes can be found in yeast—they are very ancient genes," says Gerald Rubin, the report's lead author.* "Some can be found only in worms and flies, and some only in flies. Of course the closest similarity would be with the mouse. Any gene on this list would have a parallel in the mouse."
image

Chart Legend
image
Human Areas Disease Fly Worm Yeast
Bones Multiple Exotoses
Ossification at tips of femur, pelvis, or ribs (EXT1 gene) image image image
image
Blood Leukemia
chronic myelogenosleukemia, a cancer of the blood (ABL1 gene) image image image
image
Bruton Agammaglobulinemia
lack of mature B cells (BTK gene) image image image
image
G6PD Deficiency
drug- and stress-induced rupture of red blood cells (G6PD gene) image image image
image
Brain Early-Onset Alzheimer Disease
(PS1 gene) image image image
image
Fragile X Syndrome
common cause of mental retardation (FMR1 gene) image image image
image
Juvenile Parkinson Disease
(PARK2 gene) image image image
image
Colon Hereditary Nonpolyposis Cancer
(MSH2 gene) image image image
image
Adenomatous Polyposis
polyps that become malignant (APC gene) image image image
image
Ears Hereditary Deafness
(MYO15 gene) image image image
image
Eyes Retinoblastoma
cancer of the eye (RB1 gene) image image image
image
Heart Familial Cardiac Myopathy
inherited cardiac disease (MYH7 gene) image image image
image
Long QT Syndrome
sometimes fatal cardiac arrhythmias (3-SCN5A gene) image image image
image
Kidney Polycystic Kidney Disease 2
(PKD2 gene) image image image
image
Liver Wilson Disease
buildup of copper in cells, causing liver disease and other symptoms (ATP7B gene) image image image
image
Lung Cystic Fibrosis
progressive disease of lungs and pancreas (CFTR gene) image image image
image
Lung Cancer
caused by defects in P53 gene, which can also cause cancer of esophagus, colon, brain, lung, breast, and skin (P53 gene) image image image
image
Muscles Duchenne Muscular Dystrophy
progressive atrophy of muscles (DMD gene) image image image
image
Pancreas Pancreatic Cancer
(MADH4 gene) image image image
image
Pancreatic Cancer
(RAS gene) image image image
image
Prostate Advanced Cancer of the Prostate
caused by mutations in PTEN gene, which can also cause cancer of brain, endometrium, and breast (PTEN gene) image image image
image
Skin Xeroderma Pigmentosum D
early-onset skin cancer (XPD gene) image image image
image
Neurofibromatosis 1
soft tumors at many sites, plus skeletal and neurologic defects (NF1 gene) image image image
image
Thyroid Cancer of the Thyroid
multiple endocrine neoplasia type 2 (MEN2 gene) image image image
image

* SOURCE: G.M. Rubin et al., "Comparative Geonomics of Eukaryotes," Science 287 (March 24, 2000): 2210-11, fig. 1.

Glurch Meets Oobleck

2005-03-30T19:44:00.000-08:00

Glurch Meets Oobleck

Glurch Meets Oobleck

Science Process Skills

* observing
* communicating
* comparing and measuring

Glurch Materials

* resealable quart size plastic bags
* measuring spoons
* 45 ml liquid laundry starch
* 25 ml liquid white glue
* 1/4 tsp. salt

To prepare:

1. Pour the starch into a baggie. Add salt and mix until it is completely dissolved.
2. Add the white glue.
3. Squeeze out the excess starch until the substance becomes doughy.
4. Knead. If the glurch is runny, add a few more grains of salt.

Oobleck Materials

* resealable quart size plastic bags
* measuring spoons
* 1 Tbs + 1 tsp water
* 2 Tbs corn starch
* 2 drops food coloring (optional)

To prepare:

1. Open your plastic bag and add 1 tablespoon of water.
2. Measure 2 tablespoons of corn starch and add to the water in the plastic bag. If you would like, add two drops of food coloring to the bag before sealing the bag.
3. Seal the plastic bag and mix the corn starch and water. Try shaking and kneading the plastic bag.

Doing the Activity

1. Look at the substances in your bags and make some observations about them. What do you see? What does it look like?
2. Open the top of each bag and touch the substances. What do they feel like? Does either one feel like anything that you have touched before?

Reflecting

* If both substances were poured, which would fill a container faster?
* Which substance would make a better substitute for thumbtacks?
* Which substance would be better measured in milligrams than milliliters?
* Which substance would make less noise when pulled up from a surface?
* Which substance would make a better emergency soccer ball?

Applying
What uses do you think could be for this substance? What would you name this substance?

What's Happening
Some materials don't quite fit our normal notions of solid, liquid, and gas. A suspension such as Oobleck is one. It behaves like a liquid in some ways, but it also has properties of a solid. Silly putty, quick sand, and glass are some other examples of strange substances. The molecular bonds are stronger than in a normal liquid, but not as strong as in a solid.

More Challenges

* Read the story "Bartholomew and Oobleck" by Dr. Suess.
* Create a Glurch and Oobleck Olympics. Make up a series of tests that can be tried with Oobleck and Glurch such as which one hides more easily in your hand.
* Create an invention that uses Glurch or Oobleck.

Activity Source
Fizz, Bubble, and Goo, Iowa State University Extension- Science, Engineering and Technology Youth Initiative, 32 Curtiss Hall, Ames, IA, 50011 (not for sale).

Want to live 100 years? Eat Bulgarian yoghurt

2005-02-02T19:16:00.001-08:00

Want to live 100 years? Eat Bulgarian yoghurt

Want to live 100 years? Eat Bulgarian yoghurt

By Anna Mudeva

MOMCHILOVTSI, Bulgaria (Reuters) - Lactobacillus bulgaricus sounds like a nasty infectious disease but the organism that curdles milk may be the reason Maria Shopova recently celebrated her 100th birthday.

Unaware that she may owe her longevity to the friendly bacterium, Maria grins, unveiling her two remaining teeth, and explains: "It's luck given by God".

The lively centenarian, who kept a cow until she was 80, has lived on dairy products -- yoghurt in particular -- most of her life in the picturesque mountain village of Momchilovtsi in southern Bulgaria.

The Balkan country proudly claims to have invented yoghurt and given the world the secret to a long life but its own consumption has steadily declined since the collapse of communism.

Yoghurt is slowly disappearing from the nation's table with annual consumption falling from 40 kg (88 lb) per capita, the world's highest in the 1980s, to 22 kg in 2001.

The drop has paralleled a decline in agricultural production and incomes over the past 13 years as ex-communist Bulgaria charts a difficult path towards a market economy, industry officials said.

Perhaps not coincidentally, the number of centenarians has also fallen to 187 in 2001 out of Bulgaria's population of eight million, or less than a one in every thousand, statistics show. Around 100 years earlier, the figure was four in every thousand.

YOGHURT LINKED TO LONGEVITY

Now found at supermarkets around the world, it wasn't until the early 1900s that Russian scientist Ilya Mechnikov, a 1908 Nobel Prize winner, linked yoghurt with longevity.

Mechnikov, who worked at the Paris-based Pasteur Institute, compiled statistics from 36 countries to discover more people lived to the age of 100 in Bulgaria than in any other. He attributed this to the country's most traditional food -- home-made yoghurt.

Later, numerous scientific studies in Europe, Japan and the United States proved the bacteria in yoghurt help maintain good health by protecting the human body from toxins, infections, allergies and some types of cancer.

Historians think yoghurt was part of the diet of Bulgaria's most ancient inhabitants, the Thracians, who were good sheep breeders. They say that in Thracian yog meant "thick" and urt meant "milk" and that's how the word yoghurt appeared.

Between the fourth and sixth century BC, they used to put milk in lambskin bags, which they carried about on their waists. The warmth of the body and the bags' microflora fermented it.

Some scientists think that yoghurt's predecessor was a fermented milk drink called "kumis". It was made from mare's milk by the proto-Bulgarians, a nomadic tribe who moved from Asia to the Balkans in AD 681.

Legend says that the Mongol warlord Genghis Khan used yoghurt to feed his army because of its healthy properties.

In Western Europe, it made its debut in the 16th century in the court of the French king Francis I, when a Turkish doctor cured the king's persistent stomach trouble by putting him on a Bulgarian yoghurt diet, writes professor Hristo Chomakov in his book "Bulgarian yoghurt -- health and longevity".

"The traditional Bulgarian yoghurt is a unique product because of our country's unique microclimate," said Tsona Stefanova, head of the research centre at LB Bulgaricum, a state-run company licensed to export yoghurt know-how.

"It has its own specific taste and properties. It is sour and thick so that when you turn the pot over, yoghurt sticks and does not fall," she added.

ONLY IN BULGARIA

LB Bulgaricum has a unique collection of over 700 strains of bulgaricus, which allows it to produce various yoghurt starter cultures and achieve different flavours and density.

Over the past 30 years the company has sold yoghurt know-how to more than 20 countries, including Japan, Germany, Switzerland, the Netherlands, France, the Philippines and Austria.

"Bulgaricus can grow only in Bulgaria, elsewhere it mutates," said Georgi Georgiev, manager of Lactina which deals with research and production of health food.

Georgiev said his team had found strains of bulgaricus in soil, on some trees' bark, in blossoms and even in ant-hills in Bulgaria's most environmentally clean regions such as Momchilovtsi in the southern Rhodopa mountains.

Experiments showed that a wooden stick left over an ant-hill for a while and then dipped into boiled and cooled milk would ferment it and turn it into yoghurt, as would antique silver coins, said Georgiev's assistant Nikolai Zhilkov.

A good source of vitamin B, calcium and protein, yoghurt's virtue as a health food has defied time.

Apart from having a reputation for being kind to the digestive system, it is also an excellent face cleansing mask, a soother for sunburn and douche for a thrush attack.

"Numerous researchers have shown that fermented milk has strong anti-tumour effect, which is due to its lactic acid bacteria," said Professor Akiyoshi Hosono at Japan's Shinsho University, who studies fermented milk's anti-mutagen impacts.

International food giants such as France's Danone, Swiss Nestle and Japan's Meiji Milk Products have been using friendly bacteria to produce health food known as probiotics over the past few decades.

Although local consumption may have dropped, Bulgaria is not ready to give up on its claim as the inventor of yoghurt.

Economy Ministry officials told Reuters Sofia wanted the World Trade Organisation to prevent other countries from describing their yoghurt as "Bulgarian" or "Bulgarian-style".

"It is going to be the food of the new millennium. The world is gradually getting crazy about healthy food," said Georgiev.

Want to live 100 years? Eat Bulgarian yoghurt

2005-02-02T19:16:00.000-08:00

Time to Teach - resources for teachers

2005-01-19T20:24:00.000-08:00

Time to Teach - Interactive Whiteboard resources, using powerpoint, worksheets, and booster classes to springboard children into higher grades.

tempeh factory

2005-01-18T07:25:00.000-08:00

Category -All Social Events

Bountiful Pickings for the Festive Season, All on Tours of Singapore!

With the period of festivities nearly upon us, what better way to shop for New Year goods than to join a local tour that brings you to the cheapest and the best places? And if you are entertaining visitors from abroad, or are thinking of a way to occupy your children, educational tours are a great and cost-conscious way for you to show them the interesting side of our island.

For the month of January, the Club has organised no less than four educational tours to several must-go spots around the country.

Tour 1 - Tour of Woodlands and Lim Chu Kang – 7 Jan 03

It’s nice to get away from the hustle and bustle of the everyday crowd once in a while. On 7 January, sign up with your family to join a scenic tour of Woodlands and Lim Chu Kang, where your children can experience first hand the rustic values of farm life.

In the morning, visit a Tempeh factory, one of the few left that still produces fermented beans, and a Biscuit factory, which churns out goodies like fragrant durian biscuits and many other local favourites. A short trip to a Herbal Jelly factory is next, where you can see first-hand the different stages of jelly processing. Plus, you can buy some really fresh products from all three places if you take a fancy.

After lunch, it’s time to explore some of the few farms left in Singapore. Enjoy a visit to a Hi-Tech Vegetable farm, and see how a wide variety of herbs, spices and other greens are grown the modern way. Sample the vegetables and other products for yourself and receive a free goody bag of greens with the entry fee. You and your family will really enjoy harvesting vegetables you wish to purchase! Afterwards, it’s time to visit a Chick farm to see how different types of chickens are grown. Besides the opportunity to buy some really cheap, fresh eggs, your children will enjoy the contact with cute fluffy chicks!

Date

Tuesday, 7 Jan 03

Time

9.00am to 5.00pm

Assembly Point

Lobby, Tessensohn Clubhouse

Charges*

Member/Member’s Child** $12 Guest/Guest’s Child** $17

Group Size

30 persons (min)/40 persons (max)

Closing Date

17 Dec 02

* exclude lunch

** child above 3 but below 12 years old as at date of event

Tour 2 -Tour of Western Singapore – 17 Jan 03

If you are a smart shopper, or are looking for special gifts that are good value for money, this trip must be for you!

On 17 January, seize the opportunity to visit some really interesting spots in the West. In the morning, we will take a trip down to Orchidville, the place for all varieties of potted plants that will look perfect in homes and offices. Here, a horticulture expert will demonstrate how to care for your plants and keep them in healthy bloom. Chocolate lovers will love what’s up next. A full tour of the entire production line of a Chocolate factory supplying upmarket hotels and airlines. Sample the products to your heart’s content, and buy some for family and friends.

To keep your taste buds rolling, visit a Bird’s Nest factory next to gain first hand knowledge of how this delicious product is collected and processed. You can also learn how to differentiate the genuine thing from imitation products. Take the opportunity to purchase a great-value gift hamper that makes the perfect present for relatives and business associates. To round off a productive day, take a ride to the oldest Pottery in Singapore which still boasts of a real Dragon Kiln. See how pottery is fired the old fashioned way and hunt for new tableware to grace your dining room this coming festive season.

Date

Friday, 17 Jan 03

Time

9.00am to 5.00pm

Assembly Point

Lobby, Tessensohn Clubhouse

Charges*

Member/Member’s Child** $14 Guest/Guest’s Child** $19

Group Size

30 persons (min)/40 persons (max)

Closing Date

3 Jan 03

* exclude lunch

** child above 3 but below 12 years old as at date of event

Tour 3 -Tour of Northwestern Singapore – 21 Jan 03

With the Chinese New Year coming up, it’s time to stock up on ingredients and foods that will keep for a little while longer. This educational tour will bring you to some of the best places in Singapore to buy food straight off the production line, and at great prices.

After leaving the Clubhouse, visit a Groundnut factory to see how various groundnut products are processed. Freshly packed products can be bought for home. Your next destination is a place which produces great-tasting meat. You will enjoy visiting a BBQ meat processing factory, where all kinds of products from fish floss to sliced meat are produced and put on show. Delicious fresh-grilled meat is available at an amazing price.

Next up is a Frozen Food factory that manufactures meals like Chicken Teriyaki for hotels and homes. Take the chance to sample instant meals that come in useful when you have no time to cook. Finally, take a trip to a confectionery factory where trays after wonderful trays of golden pastries are prepared. With the low prices on show, stock up on freshly baked and packed pineapple tarts and other goodies for the coming festive season.

Date

Tuesday, 21 Jan 03

Time

9.00am to 5.00pm

Assembly Point

Lobby, Tessensohn Clubhouse

Charges*

Member $14/Member’s Child** $12

Guest $18/Guest’s Child** $15

Group Size

30 persons (min)/40 persons (max)

Closing Date

3 Jan 03

* exclude lunch

** child above 3 but below 12 years old as at date of event

Tour 4 - Night Tour of Singapore – 25 and 26 Jan 03

With the Chinese New Year just days away, it’s time for a final round of the shops to get everything else you need, and what better way to do so than to join a guided tour with fellow members that brings you to the best places? Departing from the Clubhouse at night, first stroll down Chinatown, where street stalls selling Chinese New Year items await your pleasure. Enjoy your walk around – there is so much to see and buy!

Next, pay a visit to factories that operate by night in Singapore. Follow your nose and your tastebuds as you visit plants making popular foods like carrot cake, yew char kuey, fish balls, noodles and more. These are great places to buy food hot straight off the stove.

The famous Jurong Fishery Port is next on the agenda. Here, you can catch the amazing sight of live and jumping fish and seafood being unloaded from boats. See how dealers and fishmongers bargain over the latest catch, and take the opportunity yourself to select and buy the freshest seafood in Singapore. To round off an interesting night out, visit Pasir Panjang vegetable wholesale market, where you can pick up the freshest leafy greens at prices not available elsewhere.

With these four astonishing educational tours lined up, members will get to see a rare side of Singapore and pick up amazing bargains at the same time. As places are limited, sign up today to avoid disappointment.

PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

2004-12-30T00:15:00.000-08:00

PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

PHARMACEUTICAL PRODUCTION IN TRANSGENIC ANIMALS

Biotechnology Information Series (Bio-10) North Central Regional Extension Publication Iowa State University - University Extension

A New Kind of Farming
^ ^^^ ^^^^ ^^ ^^^^^^^

A new brand of farming is emerging from the research and development labs of several universities and small biotechnology companies - so new they're even changing the spelling to "pharming."

Pharming is the production of human pharmaceuticals in farm animals that is presently in the development stage with possible commercialization by the year 2000. It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987. Transgenic organisms have been modified by genetic engineering to contain DNA from an external source. The first drugs produced by this approach are about to enter clinical trials as part of the FDA review process. These transgenic animals will likely be raised by the pharmaceutical companies and will certainly be kept separate from the food supply.

Genetic Engineering
^^^^^^^ ^^^^^^^^^^^

During the 1970s, advances in DNA manipulation techniques provided a significant, economical alternative source for many drugs made of protein. Previously, these protein drugs were available in extremely limited supplies; for example, human cadavers were the source for human growth hormone, and insulin to treat diabetes was collected from slaughtered pigs.

By genetic engineering, the DNA gene for a protein drug of interest can be transferred into another organism that will produce large amounts of the drug. This technique (illustrated in Figure 1), can be used to impart new production characteristics to an organism, as well as to trigger the production of a protein drug:

1. The gene of interest is isolated on a strand of DNA.
2. DNA is cut at specific points by restriction enzymes. The enzymes recognize certain sequences of bases on the DNA strand and cut where those sequences appear.
3. The cut DNA joins with a vector, which may be a virus or part of a bacterial cell called a plasmid. The vector carries the gene of interest into the organism that will produce the protein.
4. Transformation occurs when the gene carried by the vector is incorporated into the DNA of another organism where it initiates the action desired (production of a drug, etc.).

[Figure 1]

The first successful products of the genetic engineering process were protein drugs like insulin and growth hormone. These drugs do not have to be produced by mammals to be active in mammals. An inexpensive, easy-to-grow culture of genetically engineered bacteria like the common E. coli can manufacture these protein drugs.

[Figure 2]

Other human drugs, such as tPA for blood clots, erythropoietin for anemia, and blood clotting factors VIII and IX for hemophilia, require modifications that only cells of higher organisms like mammals can provide. The higher costs of maintaining mammalian cell cultures that produce only small amounts of the drugs have been an enormous barrier to the commercial development of this type of cell culture production method.

Animal Pharming
^^^^^^ ^^^^^^^^
By genetic engineering, the DNA gene for a protein drug of interest can be transferred into another organism for production. Which organism to use for production is a technical and economic decision. For certain protein drugs that require complex modifications or are needed in large supply, production in transgenic animals seems most efficient. The farm animal becomes a production facility with many advantages - it is reproducible, has a flexible production capacity through the number of animals bred, and maintains its own fuel supply. Best of all, in most animal drug production, the drug is delivered from the animal in a very convenient form - in the milk!

Procedure

A transgenic animal for pharmaceutical production should (1) produce the desired drug at high levels without endangering its own health and (2) pass its ability to produce the drug at high levels to its offspring.

The current strategy to achieve these objectives is to couple the DNA gene for the protein drug with a DNA signal directing production in the mammary gland. The new gene, while present in every cell of the animal, functions only in the mammary gland so the protein drug is made only in the milk. Since the mammary gland and milk are essentially "outside" the main life support systems of the animal, there is virtually no danger of disease or harm to the animal in making the "foreign" protein drug.

After the DNA gene for the protein drug has been coupled with the mammary directing signal, this DNA is injected into fertilized cow, sheep, goat, or mouse embryos with the aid of a very fine needle, a tool called a micromanipulator, and a microscope (Figure 2). The injected embryos are then implanted into recipient surrogate mothers where, hopefully, they survive and are born normally.

Commercialization Issues
^^^^^^^^^^^^^^^^^ ^^^^^^
Success in creating a transgenic animal that can produce the drug is far from guaranteed. About 10 to 30 percent of mouse embryos produce transgenics, but less than 5 percent of goats, sheep, or cows do. Production of the drug is measured during lactation after the animal is raised to maturity and bred. Because of the long time periods involved and low success rates, developing transgenic animals is currently very expensive, as the dollar amounts in Table 1 indicate.

Although most protein drugs are made in milk, a notable exception is human hemoglobin that is being made in swine blood to provide a blood substitute for human transfusions. Because hemoglobin is naturally a blood protein, it is likely to be one of few exceptions to the usual method of production in milk. Furthermore, the economics of blood production are less favorable, because to recover human hemoglobin, the animal producing it must be slaughtered.

Drugs currently made by or being developed in transgenic animals are listed in Table 1. Notice that pharming is expected to increase the value of animals dramatically. In general, animal pharming is considered to be 5 to 10 times more economical on a continuing basis and 2 to 3 times cheaper in start-up costs than cell culture production methods.
[Table 1]

Drug Animal Value/Animal/Yr*
---- ------ ---------------
AAT sheep $15,000
tPA goat 75,000
Factor VIII sheep 37,000
Factor IX sheep 20,000
Hemoglobin pig 3,000
Lactoferrin cow 20,000
CFTR sheep, mouse 75,000
Human Protein C pig 1,000,000

*Current market price of the drug and supply produced by one animal.

Drug descriptions:

AAT alpha-1-antitrypsin, inherited deficiency leads to
emphysema
tPA tissue plasminogen activator, treatment for
blood clots
Factors VIII, blood clotting factors, treatment for hemophilia
XI
Hemoglobin blood substitute for human transfusion
Lactoferrin infant formula additive
CFTR cystic fibrosis transmembrane conductance
regulator, treatment of CF
Human Protein C anticoagulant, treatment for blood clots

----------------------

Regulatory and Ethical Issues
^^^^^^^^^^ ^^^ ^^^^^^^ ^^^^^^
Production of human pharmaceuticals in farm animals has many technical barriers to overcome, although most technologists agree that these technical difficulties will be easily resolved in the 1990s. As a production method, animal pharming is entirely unprecedented and is likely to undergo significant evaluation by the Food and Drug Administration (FDA). Human drugs purified from animal milk or blood are likely to require exceptional levels of safety testing before animal and human health concerns are addressed to the satisfaction of consumers.

At a more fundamental level, many people are genuinely concerned about animal welfare and biotechnology's redefinition of the relationship between humans and animals. Genetic engineering and transgenic animal research are essentially human endeavors to improve the availability, quality, and safety of drugs; to enhance human health; and to improve animal health. Animal breeding has gone on for centuries, but the ability to change the DNA of the animal brings breeding to a revolutionary new level.

DNA profiling can be used for identifying individuals and determining relationships

2004-12-27T23:58:00.000-08:00

DNA profiling can be used for identifying individuals and determining relationships

17.4. DNA profiling can be used for identifying individuals and determining relationships

We use the term DNA profiling to refer to the general use of DNA tests to establish identity or relationships. DNA fingerprinting is reserved for the technique invented by Jeffreys et al. (1985) using multilocus probes. For more detail on this whole area, the reader should consult the book by Evett and Weir (Further reading).
17.4.1. A variety of different DNA polymorphisms have been used for profiling

DNA fingerprinting using minisatellite probes

These probes contain the common core sequence of a hypervariable dispersed repetitive sequence GGGCAGGAXG, first discovered by Jeffreys et al. (1985) in the myoglobin gene (see Section 7.4.2). When hybridized to Southern blots they give an individual-specific fingerprint of bands (Figure 17.19). Their chief disadvantage is that it is not possible to tell which pairs of bands in a fingerprint represent alleles. Thus, when matching DNA fingerprints, one matches each band individually by position and intensity. Other hypervariable repeated sequences have been used in the same way, for example those detected by the synthetic oligonucleotide (CAC)5 (Krawczak and Schmidtke, 1998). top link
DNA profiling using single-locus minisatellite markers

Minisatellite probes recognize single-locus variable tandem repeats on Southern blots. Each probe should reveal two bands in any person's DNA, representing the two alleles. Profiling is based on four to ten different polymorphisms. These probes allow exact calculations of probabilities (of paternity, of the suspect not being the rapist, etc.), if the gene frequency of each allele in the population is known. For matching alleles between different gel tracks, the continuously variable distance along the gel has to be divided into a number of ‘bins'. Bands falling within the same bin are deemed to match. It is imperative that the criteria used for judging matches in each profiling test should be the same binning criteria that were used to calculate the population frequencies of each allele. The binning criteria can be arbitrary within certain limits, but they must be consistent. Minor variations within repeated units of some minisatellites potentially allow an almost infinite variety of alleles to be discriminated, so that the genotype at a single locus might suffice to identify an individual (Jeffreys et al., 1991).top link
DNA profiling using microsatellite markers

Microsatellite polymorphisms (Section 7.4.3) are based on short tandem repeats, usually di-, tri- or tetranucleotides. They have the advantages over minisatellites that they can be typed by PCR and that discrete alleles can be defined unambiguously by the precise repeat number. This avoids the binning problem and makes it easier to relate the results to population gene frequencies.top link
The use of Y-chromosome and mitochondrial polymorphisms

For tracing relationships to dead persons, Y-chromosome and mitochondrial DNA polymorphisms are especially useful because of their sex-specific pattern of transmission. An interesting example was the identification of the remains of the Russian Tsar and his family, killed by the Bolsheviks in 1917, by comparing DNA profiles of excavated remains with living distant relatives (Gill et al., 1994).top link
17.4.2. DNA profiling can be used to determine the zygosity of twins

In studying nonmendelian characters (Chapter 19), and sometimes in genetic counseling, it is important to know whether a pair of twins are monozygotic (MZ, identical) or dizygotic (DZ, fraternal). Traditional methods depended on an assessment of phenotypic resemblance or on the condition of the membranes at birth (twins contained within a single chorion are always MZ, though the converse is not true). Errors in zygosity determination systematically inflate heritability estimates for nonmendelian characters, because very similar DZ twins are wrongly counted as MZ, while very different MZ twins are wrongly scored as DZ.

Genetic markers provide a much more reliable test of zygosity. The extensive literature on using blood groups for this purpose is summarized by Race and Sanger (1975). DNA profiling is nowadays the method of choice. The Jeffreys fingerprinting probe allows a very simple test - samples from MZ twins look like the same sample loaded twice, and samples from DZ twins show some differences. An error rate could be calculated from empirical data on band sharing by unrelated people, using some defined binning strategy (see above).

When single-locus markers are used, if twins give the same types, then for each locus, the probability that DZ twins would type alike is calculated. If the parents have been typed, this follows from mendelian principles; otherwise the probability of DZ twins typing the same must be calculated for each possible parental mating and weighted by the probability of that mating calculated from population gene frequencies. The resultant probabilities for each (unlinked) locus are multiplied, to give an overall likelihood PI that DZ twins would give the same results with all the markers used. The probability that the twins are MZ is then:

where m is the proportion of twins in the population who are MZ (about 0.4 for like-sex pairs). Sample calculations are given in Appendix 4 of Vogel and Motulsky (Further reading).top link
17.4.3. DNA profiling can be used to disprove or establish paternity

Excluding paternity is fairly simple - if the child has a marker allele not present in either the mother or alleged father then, barring new mutations, the alleged father is not the biological father. Proving paternity is, in principle, impossible - one can never prove that there is not another man in the world who could have given the child that particular set of marker alleles. All one can do is establish a probability of nonpaternity that is low enough to satisfy the courts and, if possible, the putative father.

DNA fingerprinting probes have been widely used for this purpose (Figure 17.19). Bands must be binned according to an arbitrary but consistent scheme, as explained above, to decide whether or not each nonmaternal band in the child fits a band in the alleged father. Then if, say, 10/10 bands fit, the odds that the suspect, rather than a random man from the population, is the father are 1:p10, where p is the chance that a random man from that population would have a band matching a given band in the child. Even for p = 0.2, p10 is only 10-7. Single-locus probes allow a more explicit calculation of the odds (Figure 17.20). A series of four to ten unlinked single-locus markers can give overwhelming odds favoring paternity if all the bands fit.top link
17.4.4. DNA profiling is a powerful tool for forensic investigations

DNA profiling for forensic purposes follows the same principles as paternity testing. Scene-of-crime material (bloodstains, hairs or a vaginal swab from a rape victim) are typed and matched to a DNA sample from the suspect. If the bands don't match, the suspect is excluded. One of the most powerful applications of DNA profiling is for preventing miscarriages of justice. If the bands do all match, the odds that the criminal is the suspect rather than a random member of the population can be calculated, based on the allele frequencies in the population. Of course, if the alternative were the suspect's brother, the odds would look very different. The fate of DNA evidence in courts provides a fascinating insight into the difference between scientific and legal cultures. There are at least three stumbling blocks for DNA data.

* The jury may simply not believe, or perhaps choose to ignore, the DNA data, as evidently happened in the OJ Simpson trial. A fascinating account of the DNA evidence is given by Weir (1995).
* The jury may be led into a false probability argument, the so-called Prosecutor's Fallacy. Suppose a suspect's DNA profile matches the scene-of-crime sample. The Prosecutor's Fallacy confuses two different probabilities:
1. the probability the suspect is innocent, given the match;
2. the probability of a match, given that the suspect is innocent.

The jury should consider the first probability, not the second.

Using Bayesian notation (Box 17.2), with M = match, G = suspect is guilty, I = suspect is innocent, we want to calculate PI | M, and not PM | I. If the suspect were guilty, the samples would necessarily match: PM | G = 1. Population genetic arguments might say there is a 1 in 106 chance that a randomly selected person would have the same profile as the crime sample: PM | I = 10-6. Suppose the guilty person could have been any one of 107 men in the local population. If there is no other evidence to implicate him, he is simply a random member of the population and the prior probability that he is guilty (before considering the DNA evidence) is PG = 10-7. The prior probability that he is innocent is PI = 1–10-7, ~ 1. Baye's theorem tells us that

The prosecutor would no doubt be happy to see the jury use 106 instead of 0.9 for the probability that the suspect is innocent! Given the Bayesian argument, it is clear that a forensic test needs PM | I to be 10-10 or less if it is to be able to convict a suspect on DNA evidence alone.
* Objections may be raised to some of the principles by which DNA-based probabilities are calculated.
1. The multiplicative principle, that the overall probabilities can be obtained by multiplying the individual probability for each band or locus, depends on the assumption that bands are independent. If the population were actually stratified into reproductively isolated groups, each of whom tended to have a particular subset of bands or alleles, the calculation would be misleading. This is serious because it is the multiplicative principle that allows such exceedingly definite likelihoods to be given.
2. For single-locus markers, the probability depends on the gene frequencies. DNA profiling laboratories maintain databases of gene frequencies - but were these determined in an appropriate ethnic group for the case being considered? Taken to extremes, this argument implies that the DNA evidence might identify the criminal as belonging to a particular ethnic group, but would not show which member of the group it was who committed the crime.

These issues have been debated at great length, especially in the American courts. Both objections are valid in principle, but the question is whether they make enough difference to matter. General opinion is that they do not. It would be ironic if courts, seeing opposing expert witnesses giving odds of correct identification differing a million-fold (105:1 versus 1011:1), were to decide that DNA evidence is hopelessly unreliable, and turn instead to eye-witness identification (odds of correct identification < 50:50).top link

PV92 Alu-Polymorphism

2004-12-27T23:39:00.000-08:00

PV92 Alu-Polymorphism

The human genome consists to about 50% of transposable DNA elements, so-called "jumping genes". These DNA stretches constitute a major portion of our non-coding or "Junk"-DNA. Some of these elements jump, leaving one location to insert themselves into another. Others are being copied into new locations, resulting in ever growing numbers of insertions across the human genome. Whatever their mechanism, all transposable elements have the potential to affect change through mutations, duplications, or deletions.
One major transposon found in all primates is called Alu. Alu does not jump by itself but gets copied by way of reverse trancriptase encoded by another transposon, L1. Alu has emerged so early in time that it exists in all primates inserted into approximately 1,000,000 different locations across each species' genome. Thus, with the exception of roughly 2,000 human-specific insertions, most human Alu insertions can be found in their homologous positions in the genomes of other primates, too.
PV92 Alu Insertion Polymorphism detects the presence or absence of a "jumping gene" on chromosome 16. This simple genetic system has only three alleles and nine genotypes. Despite this simplicity, allele frequencies vary greatly in different world populations. Alternative explanations about the causes of this variation are consistent with opposing theories of the origins of modern humans "Out of Africa" vs. "Multiregional".

Re: Human Alu PCR Lab Report (200Pt. Due 4/15)

2004-12-27T22:49:00.000-08:00

Re: Human Alu PCR Lab Report (200Pt. Due 4/15)

Introduction The Alu element is actually a family of DNA sequences found only in primate genomes. (Dolan, 2001) There 14 known families of the Alu element and the family relationship are based on the gene sequence similarity. (Russel 1998) All 14 families are present in the human genome. The more recently evolved the organism, the more different variations of the Alu element will be present. Alu insertion polymorphism is autosomal markers that reflect both the maternal and paternal history of a population (Nasidze, 2001). They are stable markers that reflect unique evolutionary events. Alu element is a type of gene known as a polymorphic transposon. In humans there are over 1.5 million copies of the 14 subfamilies of the Alu gene with the average similarity being between 85% and 98 % (Makalowski, 1995). The transposon is flanked by inverted short terminal repeats and encode for the protein transposase which allow the gene to be inserted into the DNA (Makalowski, 1995). How an Alu element transposes: (Dolan, 2001)-First, the inserted Alu is transcribed into messenger RNA by the cellular RNA polymerase. -Then, the mRNA is converted to a double-stranded DNA molecule by reverse transcriptase. -Finally, the DNA copy of Alu is integrated into a new chromosomal locus at the site of a single- or double-stranded break. The Alu element is considered to be a class of mobile elements known as Short Interspersed Elements (Nasidze, 2001). The elements are usually about 300 base-pairs in length and are interspersed throughout the genome. For a long period it was though that the Alu element had no function at all and the true function is still unknown. Alu rarely transposes itself into regions critical for DNA function where it would possibly corrupt the code. This leads to the conclusion that the Alu gene family has the ability to modify gene for a genetic advantage. The Alu family of genes may be involved in regulating mutations to deal with stress. (Chu et al, 1998). Most Alu have both of the paired chromosomes with an insertion at the same position. However, many Alu insertion can be dimorphic, meaning that an insertion may be present or absent on each paired chromosome (Dr. Lin’s notes, 2003).

Colin Pitchfork first to be convicted w dna fingerprinting

2004-12-27T19:54:00.000-08:00

Colin Pitchfork

Colin Pitchfork - first murder conviction on DNA evidence also clears the prime suspect

Two schoolgirls who were murdered in the small town of Narborough in Leicestershire in 1983 and 1986 sparked a murder hunt that was only to be resolved by a intelligence-led screen, eventually leading to the conviction of a local man - Colin Pitchfork.

In 1983, a 15-year-old schoolgirl was found raped and murdered. A semen sample taken from Lynda Mann’s body was found to belong to a person with type A blood group and an enzyme profile, which matched 10 per cent of the adult male population. At that time, with no other leads or forensic evidence, the murder hunt was eventually wound down.

Three years later, Dawn Ashworth, also 15, was found strangled and sexually assaulted in the same town. Police were convinced the same assailant had committed both murders. Semen samples recovered from Dawn’s body revealed her attacker had the same blood type as Lynda’s murderer.

The prime suspect was a local boy, who after questioning revealed previously unreleased details about Dawn Ashworth’s body. Further questioning led to his confession but he denied any involvement in the first murder – that of Lynda Mann.

Convinced that he had committed both crimes, officers from Leicestershire Constabulary contacted Professor Sir Alec Jeffreys at Leicester University who had developed a technique for creating DNA profiles. Dr Jeffreys - along with Dr Peter Gill and Dr Dave Werrett of the Forensic Science Service (FSS) - had jointly published the first paper on applying DNA profiling to forensic science. Significantly, in 1985, they were the first to demonstrate that DNA could be obtained from crime stains, which proved vital in this case.

Dr Gill said: "I was responsible for developing all of the DNA extraction techniques and demonstrating that it was possible after all to obtain DNA profiles from old stains. The biggest achievement was developing the preferential extraction method to separate sperm from vaginal cells – without this method it would have been difficult to use DNA in rape cases."

Using this technique Dr Jeffreys compared semen samples from both murders against a blood sample from the suspect, which conclusively proved that both girls were killed by the same man, but not the suspect. The police then contacted the FSS to verify Dr Jeffrey’s results and decide which direction to take the investigation.

Peter Gill said: "Since the technique had not been used in criminal casework before, the FSS were asked by the police to confirm Dr Jeffrey’s conclusions. Accordingly, we carried out further tests and indeed demonstrated that the prime suspect could be excluded."

This suspect became the first person in the world to be exonerated of murder through the use of DNA profiling. Professor Alec Jeffreys said " I have no doubt whatsoever that he would have been found guilty had it not been for DNA evidence. That was a remarkable occurrence."

The police then decided to undertake the world’s first DNA intelligence-led screen. All adult males in three villages – a total of 5,000 men - were asked to volunteer and provide blood or saliva samples. Blood grouping was performed and DNA profiling carried out by the FSS on the 10 per cent of men who had the same blood type as the killer.

The murderer almost escaped again by getting a friend to give blood in his name. However, this friend was later overheard talking about the switch and that he’d given his sample masquerading as Colin Pitchfork.

A local baker, Colin Pitchfork was arrested and his DNA profile matched with the semen from both murders. In 1988 he was sentenced to life for the two murders.

An Interview with Sir Alec Jeffreys on DNA fingerprinting

2004-12-27T19:48:00.000-08:00

An Interview with Sir Alec Jeffreys

Sir Alec Jeffreys on DNA Profiling and Minisatellites
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Sir Alec Jefferys
"The terminology that we developed for DNA typing using multi-locus probes has been hijacked and in a very misleading way," says sir Alec Jefferys of the University of Leicester.

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GO TO: The InterviewsSince the discovery of the structure of DNA in 1953, knowledge of the composition and organization of the genetic material has accumulated at an astonishing pace. By the early 1980s it had become clear that most human DNA shows very little variation from one person to another. The small percentage that does vary presents enormous potential for fruitful study.

Sir Alec Jeffreys's involvement with mammalian molecular genetics began in 1975, when, as a postdoc, he moved from Oxford University to the University of Amsterdam to work with Dick Flavell. There, the two and their colleagues tried to clone a mammalian single-copy gene. They failed, but in the process managed to develop the Southern blot hybridization technique to the point where they could directly detect single-copy genes–and, in so doing, discovered one of the first examples of introns.

When Jeffreys moved to the University of Leicester in 1977, he chose to change direction completely and study DNA variation and the evolution of gene families. As a result of this work, his laboratory produced one of the first descriptions of RFLPs–restriction fragment length polymorphisms–a common form of variation in human DNA. The aim of the work was to develop a new breed of markers using DNA to track the position of genes. To develop good markers, the researchers needed to find highly variable regions of DNA.

In 1980, another team made one of the major breakthroughs in the study of DNA polymorphism, with their fortuitous discovery of the first "hypervariable" region of human DNA. These regions were found to consist of short tandem sequences repeated over and over again.

In 1983, Jeffreys found that these repeat sequences, dubbed "minisatellites," contain certain "core" sequences. This opened the way for the development of probes, containing the core sequences, for detecting many other such regions of variable DNA. One Monday morning in September 1984, Jeffreys and colleague Vicky Wilson successfully tested the effectiveness of such a probe. "The implications for individual identification and kinship analysis were obvious.... It was clear that these hypervariable DNA patterns offered the promise of a truly individual-specific identification system," Jeffreys wrote later (see A.J. Jeffreys, Am. J. Hum. Genet., 53[1]:1-5, 1993). They had stumbled on DNA fingerprinting, and Jeffreys's life was changed.

Jeffreys, 45, gained a first-class degree in biochemistry from Oxford University in 1972, and his Ph.D., also from Oxford, in 1975. After working in Amsterdam with Flavell between 1975 and 1977, Jeffreys moved to the University of Leicester as a lecturer in genetics and became a full professor in 1987. He was elected a Fellow of the Royal Society (FRS) in 1986.

Science Watch's European correspondent Amir Amirani
spoke with Jeffreys at his laboratory in Leicester.

SW:Your most-cited paper, "Hypervariable minisatellite regions in human DNA," appeared in Nature in 1985. Is the paper highly cited because it's subsequently been used in fingerprinting, or because of the light that the paper shed on the structure of variable DNA?

Jeffreys: The citations, I think, reflect the fact that at the time this was a novel, very powerful generalized technology that could be applied to a wide range of problems in human and nonhuman genetics. The paper described for the first time a general method for getting at large numbers of highly variable regions of human DNA. Also, almost as an accidental by-product, it suggested approaches for not only developing genetic markers for medical genetic research, but for opening up the whole field of forensic DNA typing. And, from the work in that first paper, we could see immediately the potential applications in individual identification and in establishing family relationships–for example in paternity and immigration disputes.
Although it wasn't mentioned in the paper for patenting reasons, we also saw the potential for exactly the same technology being applied to nonhuman species as well. This opened up all sorts of interesting possibilities in animal breeding, conservation biology, ecological genetics, and the like.

SW:Has the potential for the animal work been fulfilled?

Jeffreys: Very much so. That original DNA fingerprinting system, for example, has been used in a fair number of zoos to try and establish family relationships within captive colonies of endangered species of animals and birds, in particular to identify cases of close relationship–those individuals that you do not want to interbreed. The aim, in other words, is to minimize inbreeding and maintain genetic diversity.

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Sir Alec Jeffreys's Most-Cited Papers
Published Since 1985
(Citations updated through 1996)

Rank

Paper
Citations
through 6/94* Citations
through 12/96

Avg. cites per year
through 1996
1 A.J. Jeffreys, V. Wilson, S.L. Thein, "Hypervariable minisatellite regions in human DNA," Nature, 314(6006):67-73, 1985. 1,407 1,778 148
2 A.J. Jeffreys, V. Wilson, S.L. Thein, "Individual-specific fingerprints of human DNA," Nature, 316(6023):76-9, 1985. 669 857 71
3 E. Solomon, R. Voss, V. Hall, W.F. Bodmer, J.R. Jass, A.J. Jeffreys, F.C. Lucibello, I. Patel, S.H. Rider, "Chromosome 5 allele loss in human colorectal carcinomas," Nature, 328(6131):616-9, 1987. 444 485 49
4 A.J. Jeffreys, N.J. Royle, V. Wilson, Z. Wong, "Spontaneous mutation rates to new length alleles at tandem repetitive hypervariable loci in human DNA," Nature, 332(6161):278-81, 1988. 309 434 48
5 Z. Wong, V. Wilson, I. Patel, S. Povey, A.J. Jeffreys, "Characterization of a panel of highly variable minisatellites cloned from human DNA," Ann. Hum. Gen., 51:269-88, 1987. 308 375 38
SOURCE: ISI's Personal Citation Report, 1981-96
*citations reported with original interview

SW:Is there a biological function for mini- and microsatellites?

Jeffreys: That is a very, very tough question. If we look at minisatellites, by and large, there seems to be no obvious biological function. There are a few cases in the human genome, and a fair number of cases outside the human genome, of minisatellites that actually form part of genes. So there are tandem repeated DNA sequences that code for tandem repeated protein sequences. But those are the exception, not the rule. The majority of the minisatellite loci we look at have no obvious function. However, one area that we are very actively examining at the moment is the whole question of how variation arises at these tandem repeat DNA sequences. And that means exploring the mutation processes that go on in sperm and eggs, creating new versions.
What's come out of that is actually a very surprising result in which the mutation process, rather than just reflecting the instability of tandem repeat DNA, seems to be actively controlled by elements external to the tandem repeats. So it looks as though the tandem repeats themselves are not so unstable, but rather the instability is being directed from a locally acting regulator. We also know that the mutation process is astonishingly complex and operates by a process that is wholly unexpected for minisatellites. We call this process "gene conversion," and it involves chunks of DNA being shifted from one allele to another during the mutation process.
We also suspect that, in males, the majority of sperm mutations are specific to the male germline and may be meiotic in origin. This suggests a type of recombinational process, controlled by some elements near the minisatellite, and it looks as if it's meiotic as well. And that really does start raising questions–such as, maybe this mutation process isn't just some sort of accidental artifact of having tandem repeat DNA, but rather reflects some basic biological process going on in the DNA. One of our main jobs now is to explore this in a lot more detail.

SW:And is that of purely theoretical interest, or are there going to be practical implications as well?

Jeffreys: This is basic biology. As to whether there will be practical implications, I don't know. However, in the course of our investigations, we've developed various new strategies for detecting new mutations in human DNA, and this does, in principle, offer practical applications. By mutations, I don't mean, for example, a cystic fibrosis mutation, which is actually not a mutation at all but a variant that's been around for thousands of years. I'm talking about new mutations–actually catching DNA at the point where it has altered its structure. If we can develop methods for measuring mutation rate in an individual undergoing this process–and this is one of my main interests–then we can start asking basic questions about environmental agents, such as ionizing radiation, which might impact upon the mutation rate.

SW:Fingerprinting has been subject to a lot of controversy, something you have alluded to in some of your papers. Do you personally have any reservations about its reliability?

Jeffreys: Before I answer that, we must clear up a point on semantics, and this is not trivial. The original DNA fingerprinting system we developed, which for technical reasons is not that useful in forensic identification, produces patterns that are idiotypes–they are, for all intents and purposes, completely unique to an individual, except for identical twins. There's no serious dispute about that in my view. Unfortunately, the second generation of DNA typing systems–which is DNA profiling using single-locus probes–do not produce individual-specific patterns test by test. Even with a typical battery of five different tests, they produce patterns where unrelated people are most unlikely, in fact extremely unlikely, to share the same pattern. However, when you come to close relatives, brothers and sisters, there is a real chance, in fact about 1 in 4 to the power of 5 chance, of a brother and sister match, which is 1 in 1,000.
So, for every 1,000 sibling pairs, over five probes, you find a complete match. So they are not DNA fingerprints, not unique to an individual. However, their variability among unrelated people is pretty spectacular over five tests. Unfortunately–and particularly in the United States–the term "DNA fingerprinting," which we specifically apply to the original multi-locus system in which we look at scores of markers, has been corrupted to be used in almost any DNA typing system. That has created a problem in court, because DNA profiling does not produce DNA fingerprints, but if you call them DNA fingerprints, then the defense lawyer can stand up in court and say, "This is misleading," and that's quite right.
So this is a semantic problem, but a serious one. Basically, the terminology that we developed for DNA typing using multi-locus probes has been hijacked, and in a misleading way. Now, if we get rid of that semantic part, we can ask how valid is the huge amount of debate that's gone on about the reliability of DNA profiling? In the early days, in particular, there was real cause for concern. Some of the laboratories doing this work were carrying out real forensic analysis with technology that had been very poorly validated and hadn't been standardized.
I think that this issue has been largely addressed now, through quality controls, the adoption of standard operating conditions, blind proficiency trials, and so on. For DNA profiling, the real source of debate now relates to how one estimates the rarity of a set of DNA profiles out in the population, and how one presents that evidence in court. If you say that a DNA profile of a forensic sample matches a given suspect and is very rare in the population, then that, depending on the context, can be pretty damning evidence.

SW:Let's turn to your current research interests.

Jeffreys: My current interests are in exploring the basics of mutation of human minisatellites. We now know they are mutating by processes that are totally unexpected. These processes are probably of biological significance and may shed light on another fascinating area of human genetics: the whole area of triplet repeat instability diseases. These are microsatellites that go horribly unstable and cause neurological disease, such as Huntington's chorea, myotonic dystrophy, fragile x syndrome, and so on. These are basically microsatellites, which suddenly become highly unstable, increase their repeat number, become very long, and wreck nearby genes.
And again, for technical reasons, it's not easy to explore the details of the mutation process going on there, but we can explore in great detail the mutation process going on in minisatellites. We can use a whole battery of techniques that we've developed, which explore these bizarre mutation processes. It's not impossible, though far from guaranteed, that what we discover in minisatellites may actually be applicable to these inherited diseases.
One sort of science-fiction scenario would be this: let's suppose that what happens in minisatellites also applies to these unstable microsatellites. In other words, instability is conferred upon the array by flanking DNA, which, we suspect, is activating an allele for mutation. It's basically switching an allele on, perhaps by introducing some kind of DNA damage, such as a double strand break into the DNA.
Now, if that is true for these neurological diseases, and these diseases manifest because of this instability, one could conceivably think of some therapy aimed at blocking that mutation initiation. That's wild fantasy, but who knows? After all, gene therapy was fairly wild fantasy 20 years ago.
Another area in which we're very much involved is developing new approaches to DNA typing. We've been heavily involved over the last couple of years in an approach called digital DNA typing, where you get a digital readout from the DNA rather than the usual sort of band length measurements in DNA profiling. And that has first of all revealed minisatellites as by far the most variable loci in the human genome. The typical minisatellite has, for example, 100 million different alleles worldwide, and that is astonishingly variable. And that in turn may give us some rather interesting markers for studying recent events in human evolution–by looking at these allele structure and how they've changed over time, how they differ between recently split populations, and so on.

SW:The field of DNA fingerprinting is relatively new. How do you expect this technique to develop, and how do you expect DNA structure studies overall to progress?

Jeffreys: The field of DNA fingerprinting has diversified to the point of incoherence. It's no longer a single unified field. For example, back in 1987-88, when we had our first congress on DNA fingerprinting, the thing that welded it together was that everybody was playing around with minisatellites, DNA fingerprinting, and DNA profiling.
What's happened since then, of course, is the advent of DNA amplification by polymerase chain reaction, or PCR. This means, first of all, that there is little doubt that in forensic DNA typing within the next few years all the classic systems of DNA fingerprinting and DNA profiling will be totally replaced by PCR-driven systems. Such systems have their powers and their weaknesses as well–contamination and the like. But the advantage of PCR is that it offers great sensitivity, potential for automation, lower costs, and information that is much less ambiguous in terms of a DNA profiling result.
Now, what the ultimate DNA forensic typing system will be, I don't know. But to suppose that we've actually arrived there now is naive in the extreme, bearing in mind that information about PCR, or user-friendly PCR, was published only seven years ago. To pretend that we've gone from that to the ultimate DNA typing system is nonsense. There'll be other ones coming along, and that actually creates a major problem for the forensic scientist who is interested in databasing, because once you go in for very large-scale databasing of many thousands of people–you are trapped in that technology. You cannot change that technology because you've got to retype everybody in the database if you do. So the drive towards databasing, I think, is in fundamental conflict with the still rapidly evolving field of forensic DNA typing–the technology itself.
So I see all kinds of developments on the forensic front. People may actually come up with what everyone is talking about: DNA chips, oligonucleotide chips that will be used to interrogate PCR reactions. At the moment, these are not chips at all in the electronic sense. If one could, however, create a chip in which an oligonucleotide could detect and transduce the detection of a product (such as a PCR product) into an electronic signal, that would open up not just forensic typing, but DNA typing, medical diagnostics, and just about everything else one can think of.

Rare blood donor registry; need of the hour - Cover Story - Express Healthcare Management

2004-12-22T19:12:00.000-08:00

Rare blood donor registry; need of the hour - Cover Story - Express Healthcare Management

Rare blood donor registry; need of the hour

Shardul Nautiyal - Mumbai

Reena Mathews lost blood heavily during delivery and urgently required blood. A sample of her blood was sent to the blood bank for matching. The red cells grouped like O group, while her serum reacted with all O group cells available in the blood bank during cross-matching or compatibility test, making the blood bank official realise that the lady may be carrying the rare Bombay Blood group.

Experts inform that a rare genotype (blood group) of people was detected in Mumbai, a few decades back, who neither had A, AB, B or O group. This rare genotype was labelled as the Bombay Blood Group. If a Bombay Blood Group recipient is not transfused the blood of a Bombay Blood Group person, it can lead to a haemolytic transfusion reaction, which can be fatal and lead to death.

According to Dr Anand Deshpande, consultant, transfusion medicine and haematology, Hinduja Hospital, “Transfusion of ‘O’ group blood to these persons would result in immediate red cell lysis because of the presence of anti H antibodies in the serum of Bombay Blood Group patients. Therefore blood from only a Bombay Blood Group individual should be transfused to a Bombay Blood Group recipient.”

Studies reveal that this is due to the absence of the H substance (antigen) in the red cells. The absence of the H substance is attributed to the deficiency of the enzyme fucosyl transferase. The Bombay Blood Group phenotypes lack H antigen in the red cells and have anti-H in the serum.

Says Dr Maya Parihar Malhotra, blood bank in-charge, Bombay Hospital, “Family studies have shown that the Bombay phenotype, called as Oh, is due to the presence in homozygous state of a rare recessive gene.”

The precursor protein from which the blood group proteins are formed is termed as the H substance. This is bio-chemically produced by the binding of Fucose to the surface glycoproteins, the process being catalysed by Fucosyl transferase. If N-acetyl galactosamine binds to the H substance, it forms the blood group A, whereas if galactose binds to it, it forms the group B. Absence of any binding substance produces the O blood group.

Studies reveal that all human red blood cells with exceedingly rare exceptions carry the red cell H antigen. It is present in greatest amount on type O red cells and least on type A1B cells. The H antigen is an intermediate stage in the production of the A and B antigens. The individuals with the so-called Bombay phenotype are recognised with the presence of anti-H in the serum, in addition to anti-A and anti-B, as in type O persons.

Experts say that if proper blood grouping or testing practices is not followed, it can lead to people with Bombay blood group not being detected. According to Dr Mukesh Desai, haematologist, H N Hospital, “During cell grouping or routine grouping, Bombay Blood Group would be categorised as O group because they wouldn’t show any reaction to anti-A and anti-B antibodies just like a normal O group. When a cross matching with different blood bags of O group is done, then it would show cross-reactivity or incompatibility. Therefore Reverse grouping or Serum grouping has to be performed to detect the Bombay Blood group.”

“Other issues related to Bombay Blood Group is that blood is incompatible with all A, B and O donors. In routine forward grouping, this blood group would give reaction as an ’O’ blood group where as in serum grouping it would show reaction with ’O’ cells due to the presence of anti H in their serum,” says Dr Deshpande.

Most of the cases once detected are registered at Institute of Immuno-haematology (IIH)) for further studies as well as for availability of information regarding the donors of this group.

According to Dr Kanjaksha Ghosh, deputy director, IIH, “Since Bombay Blood Group is the rarest of the rare group, it is desirable to develop cryopreservation facilities for rare donor units. Every blood bank can easily maintain a rare blood type donor file from amongst their regular voluntary donors.”

“If these blood banks can borrow or exchange rare blood units in times of need, lot of problems related to rare blood groups like Bombay Blood Group can be solved. This is only possible if each blood bank has a large number of committed regular voluntary donors,” added Dr Ghosh.

“The wrong notion among people need to be dispelled that of the possibility of getting infections like HIV1 through blood donation. The public need to be informed that there is no way a donor can get such infection through blood donation,” opined Dr Ghosh.

Cell Grouping

Serum Grouping

Interpretation
Anti A

Anti B

Anti AB

A cells

B cells

O cells

+

-

+

-

+

-

A
-

+

+

+

-

-

B
+

+

+

-

-

-

AB
-

-

-

+

+

-

O
-

-

-

+

+

+

Bombay Blood Group

Diagram As shown in above diagram, cell grouping is carried out using anti A, anti B and anti AB commercially available sera. Serum grouping is carried out using A cells, B cells and O cells.

blood and vampires

2004-12-21T18:25:00.000-08:00

Satantra's Cathedral

Vampires in Literature by Sarah Abrahams
Vampire: Historical Origins

The image of the vampire has a long and complicated history. Many different cultures have a notion of a vampire-like character within their respective mythologies. In order to understand a modern critique of the vampire as a figure representing an array of subverted meanings, it might first be appropriate to provide a summary of vampire figures in a variety of cultural perspectives, noting both their differences and similarities. The vampire image, in a more basic sense, represents our human preoccupation with death, blood and darkness. With the advent of modern science, many of the mystical beliefs surrounding blood were abandoned. "Blood was the sight of death" (Leatherdale 16). "The concept of the vampire is founded upon two precepts: the belief in life after death, and the magical power of blood" (Leatherdale 15). The connection between the mysticism of blood and death is one founded upon observation. "Blood was the sight of death" (Leatherdale 16).
Blood

Blood has throughout history served many purposes. It was believed that both "human strength and health resided within blood" (Leatherdale 16). Blood had the power to sustain life as well as "[consuming] [it] could restore, rejuvenate, bring back life" (Leatherdale 17). The connection between blood and life is a complicated one. Blood is present in female menstruation, a symbol of the transition into adulthood, as well as in the breaking of the hymen. Blood is also present in the birth of a child. Clearly this has significance within the vampire legend.
The Living Dead

Along with notions of the magical properties of blood lied a fear in the 'living dead.' As humans have known that death is inevitable, the land of the dead seemed to be a place apart, governed by its own customs and laws of nature, which the living could not penetrate but to which they were inexorably drawn in the course of time. In certain periods of history, especially periods of disease (the plague, and more recently AIDS) humans became even more aware of the presence of death within their own communities. Because in some respects, death is the inexplicable other, humans seem to have to formed a sort of dualism where the living and the world in which they operate appears on one side and the dead the world in which they operate appears on the other.
The near universally-held belief in these two supposed laws of nature-the rejuvenating power of blood and the presumption of life after death-meant that the product of their combination (the vampire) was equally universal. The presence of the vampire can be located within a wide array of cultures.
The Transcultural Presence of the Vampire

* ·Chinese tales spoke of blood-sucking creatures that were green, covered with mold, and which had a propensity to glow in the dark.
* The Melanesian talamaur-known as the soul of the dead which preyed on the ebbing vitality of the dying.
* In India, Kali is revered as a blood-sucking mother goddess of disease, war, and death. Siva is identified with ghoulish (flesh-eating) propensities.
* In Africa, the Ashanti's asambosam, likes to suck blood through the thumbs of the sleeping. Rather than feet, asabosam stands on a pair of books.
* West Indies lore contains the loogaroo (from the French expression for werewolf- loup garou) who, disguised as an old woman, in a pact with the devil, sheds her skin and reforms as a blob of light in order to draw blood .
* A jaracara, in Brazil, resembles a snake and seeks either blood or breast milk.
* In Europe, the vampire concept seems to have developed from idea of the succubus-a female entity who would seduce young men in their sleep and "withdraw their vital fluids at the moment of peak distraction during climax." The incubus, the male counterpart associated with the devil, "would impregnate suitable female victims, such as witches" (Leatherdale 20). It is interesting to note here that only the female vampire acts out the withdrawal of essential liquid. The male vampire only serves to assist in the creation of new demonic creatures.

Though I will focus on the European history and literature of the vampire, there seems to be vampiric figures (in some form) in the folklore or religion of almost every culture.
The Vampire in Victorian Literature

In the Victorian period, the image of the vampire became a popular one. Bram Stoker's Dracula, published in 1897, most centrally explored the idea of vampirism as an interesting and complex image within literature. From Stoker's work came the figure of Dracula, who's continued portrayal in literature and film is proof of transcendence from mere literary figure to cultural icon. The Bronte's Jane Eyre and Wuthering Heights both make direct allusion to the vampire. In Jane Eyre particularly, the character of Bertha Mason is described with reference to vampirism.
Vlad Tepes- Vlad The Impaler

There has been much speculation over the creation of the vampire Dracula. Historicists have discovered the life of Vlad Tepes (Vlad the Impaler) as a potential inspiration for Bram Stoker's work and other European vampire lore. Bram Stoker occupies a large portion of Dracula to a detailed description of Romania and the conflicts surrounding the Turks. The story presented by Stoker is Vlad Tepes', other similarities can be located elsewhere in Tepes' military flair for violence and the absolutely gory nature of his destruction of his country's enemies. One scene depicted by artists, show Vlad the Impaler seated and dining among the skewered corpses of his enemies. Tepes' connections to the character of Dracula, though, are otherwise difficult to discern, though his political exile, like Dracula's societal exile, may be another area of potential similarity.
Representations of Vampirism and the Critics

There has been a long history of critical exploration in attempts to undercover the symbolic significance of the vampire. In terms of the girlhood narrative and issues of women in literature, three main themes appear most prominent and most often, all of which are based in a form of psychoanalytic theory.

Vampirism has long had associations with female sexuality. In Bram Stoker's Dracula, for example, Dracula's vampiric women epitomize the threat/fear of unleashed female sexuality. In the Victorian period, specifically with the appearance of what was called the "New Woman" (Victorian women who were more financially and emotionally independent from their families and men), the dangers of female sexuality became an issue of urgency. Traditionalists of the period worried that with women's newfound independence, these women would be more likely to explore their sexuality in ways that were considered inappropriate for women.

When one examines the specifics of dangerous female sexuality, one notices a fear of excess. The possibility of women having the potential to experience an eroticized way of life and a sexuality not limited to genital contact becomes extremely dangerous to patriarchal power. The ability for women to enact a polymorphous type of sexuality enables them, and empowers them. In order for patriarchal systems to remain in control, they insist on labeling and attempting to teach society that this form of sexuality is unhealthy and abnormal. Indeed, much of the image of the vampire deals with issues of taboo nature.

The second theme most often discussed in recent literature criticism of works containing vampire (or vampire-like) figures is the notion of gender inversion and homoeroticism. Much has been speculated with reference to Bram Stoker's Dracula, as to the autobiographical nature of the work considering the connection of the Oscar Wilde trial (with which Bram Stoker was quite close) and Dracula's creation. Considering the nearly simultaneous occurrence of the two, it seems logical to examine the vampire as a possible figure of homosexuality. Following Freud's theories, some have argued for the vampire as a sort of infant, stuck permanently in the oral phase and forced to attempt adult sexuality through it.

Images of incest are another theme of criticism of vampires in literature. In Ken Gelder's Reading the Vampire, he explains this psychosexual reading as an "ambivalent impulse of the child towards its mother" (Gelder 68). If one accepts the idea that Bram Stoker's Dracula can be interpreted through a reading of the psychosexual, the interpretations made by critics such as Twitchell, Astle, and Jackson all support the "re-enactment of that killing of the primal father who has kept all the women to himself" (Gelder 68). In Phyllis A Roth's piece "Suddenly Sexual Women in Bram Stoker's Dracula," this intepretation is extended to include what Roth explains as "the hatred of the mother"-"the Oedipal rivalry among sons and between the son and the father for the affections of the mother" (Roth 60).

All three themes reiterate one central theme-the vampire as highly sexual. In terms of Jane Eyre, one can see how Bertha's description as vampire-like makes sense considering her punishment for "uncivilized" and unladylike sexuality. In Wuthering Heights also, as a symbol of the undead, the vampire image appears in reference to Catherine, the ghost who haunts Heathcliff for his passionate cruelty.
Vampires in Modern Fiction

The vampire continues to be a popular image in fiction. The popularity of Anne Rice's Vampire Chronicles have spawned a group of modern day vampire groupies who gather near Rice's home in New Orleans. In relation to gender, Rice interprets the vampire as a definite figure of homosexuality. Within a personal and bibliographical context, it has been said that Rice's Vampire Chronicles were the result of the loss of a child. In dealing with her grief, she became obsessed with ideas of the undead and of the possibilities of eternal life. In Salem Lot, Stephen King continues the vampires in literature tradition. In King's work, though, he acknowledges the current popularity of the horror genre by telling the tale of a group of boys in modern America. As a small American town, King uses Salem's Lot as a site of particular vulnerability to the threat of vampires. Its isolation from larger society makes it an easy target.
Conclusion

As one uncovers the web-like variety in vampire related criticism, it becomes more and more clear that the reason the vampire image has remained so powerful is in it's ability to transcend a single meaning. In this way, the vampire's fluidity and multifaceted nature allows malleability in interpretations that fit all different sorts of critical thought. Most recently, in the wake of the AIDS epidemic and the gay rights movement, vampires have symbolized (for some) homosexual desire at its most potentially dangerous. Yet, when analyzed within the larger framework of vampire criticism, this seems to be only a part of a developmental and historical process of naming the vampire as symbolic of a variety of societal issues. It continues to be unknown what shape vampire criticism will manifest itself next-and it is this difficulty (or ability) of the vampire figure which continues to keep interest alive (or in vampiric terms, to remain undead).
Bibliography

* Auerbach, Nina. Our Vampires, Ourselves. Chicago: U of Chicago P. 1995.
* Botting, Fred. Gothic. from The New Critical Idiom Series. John Drakakis, ed. London: Routledge, 1996.
* Carter, Margaret L.. ed. The Vampire in Literature: A Critical Bibliography. Ann Arbor: UMI Research Press, 1989.
* "Stoker's Vampire of the Mind." in Specter or Delusion? The Supernatural in Gothic Fiction. Ann Arbor: UMI Research Press, 1987.
* Clemens, Valdine. "The Reptilian Brain at the Fin de Siecle: Dracula" The Return of the Repressed: Gothic Horror from The Castle of Otranto to Alien. NY: State University of NY Press, 1999.
* Craft, Christopher. "Kiss Me With Those Red Lips: Gender Inversion in Bram Stoker's Dracula." in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Frayling, Christopher. "Haemosexuality" in Vampyres: Lord Byron to Count Dracula. London: Faber and Faber, 1992.
* Gelder, Ken. Reading The Vampire. London: Routledge, 1994.
* Glover, David. "Sexual Aeternitatis" in Vampires, Mummies and Liberals: Bram Stoker and the Politics of Popular Fiction. Durham: Duke U P, 1996.
* Hendershot, Cyndy. "Vampire and Replicant: The One Sex Body in a Two-Sexed World." Science Fiction Studies 22 (1995) 373-98
* Leatherdale, Clive. Dracula: The Novel and the Legend. A Study of Bram Stoker's Gothic Masterpiece. Northamptonshire: Aquarian Press, 1993.
* Putner, David. "Dracula and Taboo" in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Roth, Phyllis. "Suddenly Sexual Women in Bram Stoker's Dracula" in Dracula: Contemporary Critical Essays. Glennis Byron, ed. NY: St. Martin's P, 1999.
* Schaffer, Talia. "A Wilde Desire Took Me: The Homoerotic History of Dracula" ELH 61 (1994) 381-425.
* Senf, Carol. "Dracula: Stoker's Response to the New Woman" in Victorian Studies 26:1 Autumn 1982.
* The Vampire in Nineteenth-Century English Literature. Bowling Green: Bowling Green State U Popular Press, 1988.
* Showalterm Elaine. Sexual Anarchy: Gender and Culture at the Fin de Siecle. New York: Viking- 1990
* Spear, Jeffrey L. "Gender and Sexual Dis-Ease in Dracula" in Virginal Sexuality and Textuality in Victorian Literature. Lloyd Davis, ed.. NY: State Univ of NY Press, 1993.
* Wolf, Leonard. Dracula: The Connoisseur's Guide. NY: Broadway Books, 1997.

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Red Gold | PBS

2004-12-21T02:16:00.000-08:00

Red Gold | PBS

biometric

2004-12-20T22:25:00.000-08:00

Brain Fingerprinting - Ruled Admissable

2004-12-20T21:48:00.000-08:00

Brain Fingerprinting - Ruled Admissable

Brain Fingerprinting Testing Ruled Admissible in Court

On March 5, 2001 Pottawattamie County, Iowa District Court Judge Tim O'Grady ruled that Brain Fingerprinting® testing is admissible in court. Dr. Farwell conducted a Brain Fingerprinting test on Terry Harrington, who is serving a life sentence in Iowa for a 1977 murder. The test showed that the record stored in Harrington's brain did not match the crime scene and did match the alibi. Harrington filed a petition for a new trial based on newly discovered evidence, including the Brain Fingerprinting test. On February 26, 2003 the Iowa Supreme Court reversed his murder conviction and ordered a new trial. The Iowa Supreme Court left undisturbed the law of the case establishing the admissibility of the Brain Fingerprinting evidence.

In a Brain Fingerprinting test, words, pictures or sounds describing salient features of a crime are presented by a computer, along with other, irrelevant information, that would be equally plausible for an innocent subject. Items are chosen that would be known only to the perpetrator and to investigators, but not to the public or to an innocent suspect. The subject is told which features he will see (e.g., the murder weapon), but is not told which item is correct (e. g, gun, knife, or baseball bat). When a subject recognizes something as significant in the current context, the brain emits a specific brain response. If the record of the crime is stored in the subject's brain, this response appears when the subject recognizes the correct, relevant items. If not, then the response is absent. A computerized mathematical analysis of the data determines whether or not the subject has knowledge of the salient details of the crime.

Just as a personal computer emits a characteristic sound whenever its central processing unit is transferring information to or from or the hard drive, the human brain emits a characteristic electrical brain wave response, known as a P300 and a MERMER (memory and encoding related multifaceted electroencephalographic response), whenever the subject responds to a known stimulus. The P300 electrical brain wave response, one aspect of the larger MERMER response discovered and patented by Dr. Farwell, is widely known and accepted in the scientific community. There have been hundreds of studies conducted and articles published on it over the past thirty-plus years. The MERMER, a longer and more complex response than the P300, comprises a P300 response, which is electrical events occurring 300 to 800 milliseconds after the stimulus, and additional data occurring more than 800 milliseconds after the stimulus. While a P300 shows only a peak electrical response, a MERMER has both a peak and a valley.

In order to be admissible under the prevailing Daubert standard, the science utilized in a technology is evaluated based on the following four criteria: (The Iowa courts are not bound by the Daubert criteria used in the federal courts, but they do use them when determining the admissibility of novel scientific evidence.)

1. Has the science been tested?
2. Has the science been peer reviewed and published?
3. Is the science accurate?
4. Is the science well accepted in the scientific community?

The judge ruled that Brain Fingerprinting testing met all four of the legal requirements for being admitted as valid scientific evidence. The ruling stated: "The test is based on a 'P300 effect.'… "The P300 effect has been studied by psycho-physiologists…The P300 effect has been recognized for nearly twenty years. The P300 effect has been subject to testing and peer review in the scientific community. The consensus in the community of psycho-physiologists is that the P300 effect is valid…."

The judge also ruled that "The evidence resulting from Harrington's 'brain fingerprinting' test… is newly discovered" and "material to the issues in the case," and thus meets the standard for being considered in a petition for a new trial.

The Brain Fingerprinting test on Harrington showed that the record stored in his brain did not match the crime, and did match his alibi. This is similar a finding that Harrington's fingerprints or DNA did not match the fingerprints or DNA at the crime scene, and did match those at the scene of the alibi.

Dr. Farwell conducted two different analyses of the data on Harrington. Both yielded the same conclusion. He performed the test and the analyses in strict accordance with the P300 science that has been extensively researched and is well accepted in the scientific community. In another analysis, Dr. Farwell used more state-of-the-art techniques, including the MERMER, which, though arguably more accurate, do not yet have the same level of acceptance as the P300.

After obtaining the results of the Brain Fingerprinting test, Dr. Farwell located the only alleged witness to the crime, Kevin Hughes. When Dr. Farwell confronted him with the Brain Fingerprinting test results exonerating Harrington, Hughes admitted that he had lied at Harrington's trial. He stated under oath that he had made up the story about Harrington committing the crime to avoid being prosecuted himself. Harrington’s attorney used Hughes' recantation along with Brain Fingerprinting test findings as evidence in his post-conviction petition for a new trial.

The Harrington case was about as difficult a case as could be envisioned for the Brain Fingerprinting system. The day after the crime, the perpetrator knows all about the crime and an innocent suspect knows nothing. Scientists could have readily constructed a Brain Fingerprinting test to distinguish between the two, if the technique had been invented at the time. Later, in his trial, Harrington was exposed to extensive information about the crime. This made it difficult after the trial to produce salient features of the crime that he would know only if he had committed the crime. Twenty-three years after the crime was committed, through examination of court documents, police reports, witness interviews, crime-scene photos and an investigation of crime scene itself, Dr. Farwell was able to structure a Brain Fingerprinting test that tested Harrington's brain for evidence of salient features of the crime, features that he claimed not to know because he was not there. The test showed that Harrington's brain in fact did not contain a record of these salient features of the crime. A second test conducted by Dr. Farwell showed that Harrington's brain did contain the details of his alibi.

As with other scientific evidence, Brain Fingerprinting testing does not prove guilt or innocence per se. It provides information about what is stored in the suspect's brain. A judge or jury can utilize this information in making the legal determination of guilt or innocence. The weight of the Brain Fingerprinting test evidence will be evaluated along with other evidence by a higher court in their consideration of Harrington's appeal. If a higher court finds that this and other evidence probably would have changed the result of the original trial if it had been known at the time, then Harrington will be granted a new trial.

"I believe the court’s admission of Brain Fingerprinting test results into evidence is a landmark in forensic science," said Dr. Farwell. "Innocent persons have a new technology to aid in their exoneration, and law enforcement has a new weapon with which to convict perpetrators. In the future we can use this technology to find out the truth early in a case. This will save innocent suspects from a traumatic investigation and trial, and potentially from false conviction and punishment. By allowing law enforcement to focus on the actual perpetrators, it will also save time and money in law enforcement.”

"I believe that in time we will be able to virtually eliminate false convictions through Brain Fingerprinting tests and other scientific technologies such as DNA and fingerprints. This new scientific technology will also allow us to significantly increase the number of actual criminals brought to justice."

Since the hearing, Dr. Farwell and Sharon Smith of the FBI have published their research on the MERMER in the prestigious peer-reviewed Journal of Forensic Sciences, January 2001.

Hundreds of scientific studies on this technology from dozens of laboratories have been published in the peer-reviewed scientific literature. Dr. Farwell has applied the technique not only in rigorous laboratory studies but also in over 100 real-life cases. Dr. Farwell and then FBI scientist Dr. Drew Richardson used the Brain Fingerprinting system to detect with 100% accuracy which people in a group were FBI agents and which were not by measuring brain responses to items only an FBI agent would recognize. Brain Fingerprinting testing was also 100% accurate in three studies Dr. Farwell conducted for a US intelligence agency and for the US Navy.

The Brain Fingerprinting system tests for knowledge of salient features of a crime stored in the brain. Scientists know that we don't remember everything, but we do remember significant features of major events — like committing a serious crime. By scientifically determining what is stored in a suspect's brain, Brain Fingerprinting testing provides evidence that can be used by judges and juries in making a determination as to whether the suspect committed the crime or not.

The History of Fingerprints

2004-12-20T01:45:00.000-08:00

The History of Fingerprints

What is Higher Order Thinking?

2004-12-15T02:32:00.000-08:00

What is Higher Order Thinking?

Bloom's Taxonomy of Thinking Skills

2004-12-15T01:57:00.000-08:00

Bloom's Taxonomy of Thinking Skills

Different learning styles

2004-12-15T01:56:00.000-08:00

Different learning styles

Problem Based Learning

2004-12-15T01:49:00.000-08:00

Problem Based Learning: "Problem Based Learning"

Problem Based Learning

If asked, most educators would agree that one essential goal of education is the development of students who are effective problem solvers for the Information Literacy Age. Most reports, such as the national SCANS (Survey of Necessary and Comprehensive Skills) and Goals 2000 documents, recommend such instruction. Most school goal statements allude to the need for critical thinking and problem solving skills. Recent California Frameworks in Mathematics and Science reflect consensus on this educational goal. But often such instruction in problem solving takes the approach of teaching models to students to apply to neat case studies rather than the messy problems of a real world.

Research indicates that critical thinking and problem solving skills are not typically addressed in the classroom. A number of studies indicate that in the typical classroom, 85% of teacher questions are at the recall or simple comprehension level. Questions that elicit synthesis and evaluative skills of thinking are rarely asked. The media portrays teachers as asking such simple, mindless questions in movies such as "Ferris Bueller's Day Off" and "Dead Poet's Society".

In Problem Based Learning (PBL) environments, students act as professionals and confront problems as they occur - with fuzzy edges, insufficient information, and a need to determine the best solution possible by a given date. This is the manner in which engineers, doctors, and, yes, even teachers, approach problem solving, unlike many classrooms where teachers are the "sage on the stage" and guide students to neat solutions to contrived problems.

What is Problem Based Learning?

Problem Based Learning is a curriculum development and delivery system that recognizes the need to develop problem solving skills as well as the necessity of helping students to acquire necessary knowledge and skills. Indeed, the first application of PBL was in medical schools which rigorously test the knowledge base of graduates. PBL utilizes real world problems, not hypothetical case studies with neat, convergent outcomes. It is in the process of struggling with actual problems that students learn both content and critical thinking skills.

Problem based learning thus has several distinct characteristics which may be identified and utilized in designing such curriculum. These are:

1. Reliance on problems to drive the curriculum - the problems do not test skills; they assist in development of the skills themselves.
2. The problems are truly ill-structured - there is not meant to be one solution, and as new information is gathered in a reiterative process, perception of the problem, and thus the solution, changes.
3. Students solve the problems - teachers are coaches and facilitators.
4. Students are only given guidelines for how to approach problems - there is no one formula for student approaches to the problem.
5. Authentic, performance based assessment - is a seamless part and end of the instruction.

(Adapted from Stepien, W.J. and Gallagher, S.A. 1993. "Problem-based Learning: As Authentic as it Gets." Educational Leadership. 50(7) 25-8 and Barrows, H. (1985) Designing a Problem Based Curriculum for the Pre-Clinical Years.

Problem Based Learning assists students to solve problems by the process of continually encountering the type of ill-structured problems confronted by adults or practicing professionals. As with information literacy, PBL develops students who can:

* Clearly define a problem
* Develop alternative hypotheses
* Access, evaluate, and utilize data from a variety of sources
* Alter hypotheses given new information
* Develop clearly stated solutions that fit the problem and its inherent conditions, based upon information and clearly explicated reasoning

Students with such ingrained skill are well prepared for occupations which rarely have a supervisor who has time, inclination, or knowledge to tell the worker what to do. They are also well prepared for the explosion of knowledge which gluts the world today.

Stages in Problem Based Learning

In the PBL curriculum, one may note three distinct phases of operation by students. Whether gathering knowledge through a variety of sources on the Internet, through print sources, or by speaking with experts, these stages explicated below are characteristic of PBL. Each step in the process is "hot linked" to a sample lesson developed by a SCORE Teacher on Assignment.

Stage 1: Encountering and Defining the Problem

Students are confronted with a real world scenario through authentic looking correspondence. Students may be asked to present to the Ancient World Architectural Review Board regarding their perspective about how and why great ancient monuments were built. They may ask some basic questions such as :

* What do I know already about this problem or question?
* What do I need to know to effectively address this problem or question?
* What resources can I access to determine a proposed solution or hypothesis?

At this point, a very focused Problem Statement is needed, though that statement will be altered as new information is accessed and understood.

Stage 2: Accessing, Evaluating and Utilizing information

Once they have clearly defined the problem, students might access print, human, or electronic information resources. In the case of the Southern Illinois Medical School, professors may be interviewed or medical texts examined. In the case of a city plan, calls to human resources such as the town manager or staff engineers might be of use. The Internet can be a focal point of research when a problem is constructed with that purpose. In the case of the sample problem, students may find a rich diversity of perspectives and resources preparatory to phase 3. Part of any problem is evaluation of the resource. How current is it? How credible and accurate is it? Is there any reason to suspect bias in the source? When utilizing the information, students must carefully appraise the worth of the sources they have accessed. If evaluating sites which theorize about these monuments and how and why they were built, students must carefully note and evaluate the accuracy and credibility of information posted at that site.

Stage 3: Synthesis and Performance

In this stage, students construct a solution to the problem. Students may create a multi-media production, a presentation to a body such as the U.N. Commission on Human Rights or the Ancient World Architectural Review Board, or a more traditional written paper focused around an essential question. In all cases, the students must re-organize the information is new ways. This is unlike an assignment which asks them to " make a report about the Palestinians and Israelis." This latter leads to use of the Internet as if it were a giant cyberspace encyclopedia. An assignment which asks students to propose a solution to the conflict between the Palestinian people and the Israelis involves a question which forces re-organization of information and consideration of perspectives.

Problems in Implementation

Cultural change is required to implement PBL. Students trained in the more traditional model of teaching, which features the teacher as "sage on the stage" and disseminator of knowledge, will experience culture shock of a sort. Students will wish to know expectations for a high grade. Though constructing a rubric with a teacher may allay fears, there is initial suspicion of the new approach.

Students must also learn to be part of the group. As with real life tasks, one person cannot conduct all research and make the entire presentation of the problem solution. Complaints about "hitchhikers" (those in the group who do not pull their own weight) will be heard from hard working students and their parents.

Teachers also experience major adjustments. More preliminary work must be done to design the problem and to ensure that there are enough materials available (in print, online, and through human resources) for this resource's ravenous approach. They must learn to construct problems that assist students to learn appropriate skills and knowledge. And they must learn to facilitate, rather than direct, student learning.

The Rewards

Though change from a teacher-centered to a problem and project based environment causes discomfort, those that have made the transition speak of new energy and enthusiasm for their classes. Students praise challenging tasks that prepare them for learning. For more information, see the Problem Based Learning online resources below:

* The University of Delaware has numerous articles about PBL including teaching art, science, and other courses. A good teacher resource. http://www.udel.edu/pbl/
* Howard Barrows, Southern Illinois School of Medicine (A medically focused analysis of PBL.) http://www.pbli.org
* Illinois Math and Science Academy (Includes K-12 applications in various disciplines.) http://www.imsa.edu/

If you have further questions about PBL, please email Bob Benoit of the Butte County Office of Education at bbenoit@bcoe.butte.k12.ca.us. Bob has directed a PBL project which included six high schools and 30 teachers over the last four years.

A Selected Problem Based Bibliography
Books:

* Barrows, H. (1994) Practice-Based Learning: Problem-Based Learning Applied to Medical Education. Springfield, Il: Southern Illinois University School of Medicine
* Barrows, H. (1985) Designing a Problem Based Curriculum for the Pre-Clinical years. New York: Springer Publishing Company.
* Boud, D., Felleti, G. (1991) The Challenge of Problem-Based Learning. London: Kogan.
* Woods, Donald R. (1994). Problem-Based Learning: How to Gain the Most from PBL. Hamilton, Ontario, Canada. Donald R. Woods, Publisher.

Selected Articles

* Barrows, Howard. See Southern Illinois University School of Medicine Homepage for an extensive list of articles published in medical journals.
* Gallagher, S., Rosenthal, H., and Stepien, W. (1992) "The Effects of Problem-Based Learning on Problem Solving. Gifted Child Quarterly, 36(4), 195-200.
* Knoll, Jean W. (1993). "An Introduction to Reiterative PBL." Issues and Inquiry in College Learning and Teaching. Spr/Smr. 19-36
* Stepien, W. and Gallagher, S., and Workman, D. (1993) "Problem-Based Learning for Traditional and Interdisciplinary Classrooms." Journal for the Education of the Gifted, 16(d4), 338-357.
* Stepien, W. and Gallagher, S.A. (1993). "Problem-based Learning: As Authentic as it Gets." Educational Leadership. 50(7), 25-8

DNA Extraction from vege and humans

2004-12-12T18:22:00.000-08:00

Nexus Research Group - FUN Science and home experiments...: "To isolate plant DNA

* blend 50g of Cauliflower in 200 mL of water for 30 seconds to get single cells.
* Strain through muslin cloth. Pour 15 mL of liquid into a screw cap tube with 3 drops of detergent and shake gently for 5 seconds.
* Carefully layer to top of tube with cold alcohol (or meths). Use a hooked glass rod to spool out strands of DNA at the interface.

To isolate your own human DNA

(an original protocol independantly developed by Nexus, please acknowledge our contribution if using this protocol for teaching purposes)

* To a 1/2 cup of water dissolve about 1/2 teaspoon of salt and add a squirt of dish-washing detergent. Save this for step 3
* Swirl about 25 mls of water around your mouth for 30 seconds. This removes some cheek cells. Spit into a disposable cup
* Add about 2cm of the fluid to a test-tube (or a Fuji film cannister) and add about 1cm of the saline/detergent solution. Invert gently 3 or 4 times to mix well (but you don't want a lot of froth). This will break open the many cheek cells you spat out, releasing the DNA message that each cell must carry.
* Layer on top some ice cold ethanol (or methanol). Strands of DNA will be seen where the two layers meet. Hook out the strands of DNA that form with a glass hook (or one made from a plastic twist-tie) by slowly dipping up and down through the two layers.

CHEMICAL TESTS TO CONFIRM THE PRESENCE OF DNA:-

The following chemical tests can be used to check you actually have DNA:

* Test for purines: Add excess 2M ammonia solution and a few drops of 0.1M silver nitrate to 1 mL of DNA extract. A white precipitate indicates the presence of purines.
* Test for phosphate: Add 1 mL of 0.2M ammonium molybdate (39.02g/L) to 0.5 mL of extracted DNA and warm gently at 60-70oC. DO NOT BOIL. Yellow colour indicates presence of phosphate.
* Test for deoxyribose: Add 2 mL of Disches reagent to 1 mL of extracted DNA. Boil in water bath for 15 minutes. Green-blue colour indicates presence of deoxyribose. Disches reagent: 486 mL glacial acetic acid, 14 mL conc. sulphuric acid, 5g diphenylamine. Stir well and add 500 mL distilled water"