DNA Transformation in Bacteria

DNA Transformation in Bacteria 1.0 Introduction and Objectives The ability of bacteria to incorporate DNA from external sources is the primary reason for their survival and proliferation. Bacteria can take DNA from their surroundings or from other bacterial cells by cell wall-transfer. While an interesting phenomenon to examine for scientists, practically it is of great concern for the human race and a source of constant challenge for the Pharmaceutical Industry. The ability of bacteria to modify their genetic information has given rise to problems such as antibiotic resistance wherein bacteria become resistant to medications that were once effective in eliminating them. In this experiment, we examine the development of antibiotic resistance in bacteria. Circular DNA called plasmids are introduced in bacteria whose cells have been modified to promote uptake of plasmid DNA. This plasmid DNA will give rise to antibiotic resistance in the bacteria, which can be observed by allowing the bacteria to proliferate in an environment containing the antibi otic. Modification of genetic information in bacteria may be a source of concern, but that ability in the hands of humans has always been coveted. Genetic engineering is an increasingly popular research area given the breakthroughs made in recent years and the potential for commercial application. Various applications require large quantities of specific DNA sequences and this is where the bacterial ability to uptake DNA and reproduce it is beneficial. Introducing plasmids containing desired sequences into bacteria, allowing bacteria to reproduce and then isolating the required DNA is a common method used to obtain large quantities of particular DNA sequences. This aspect is also explored in this experiment. 1.1 Objectives The objectives of this experiment are to: a) Observe and examine the phenomenon of DNA Transformation. b) Observe the development of antibiotic resistance in bacteria through the process of gene transformation. c) Inculcate proper Sterile Technique for laboratory procedures involving bacterial strains. 2.0 Principles This section explores the underlying concept behind the experiment. Genetic Transformation is a process of horizontal gene transfer whereby DNA from the environment is taken up by a host cell. In this experiment bacterial cells are transformed. Escherichia Coli bacteria, which are generally non pathogenic are used in this experiment. The plasmids which constitute the external DNA contain a gene that makes the cell ampicillin resistant. Ampicillin is a bacteriostatic and will normally prevent the reproduction of E. Coli bacteria. This provides us with an easy way to test if gene transformation has occurred and to what extent by means of calculating the transformation efficiency. The introduction of genetic material within the bacterial cell is done by the process of electroporation. Electroporation involves applying an electrical voltage across the bacterial cells containing the plasmids. The ionic concentration of the DNA is kept low to prevent arcing. When the voltage is applied, holes open up in the walls of the bacteria. The plasmids can then enter the bacterial cells through these holes. Application of the voltage is done for a very short period of time. As soon as electric current stops flowing, the holes in the cell wall begin to close. A nutrient rich medium is then added to the bacterial cells, some of which will have transformed, to aid cell recovery. Incubation is then carried out, after which the cell suspension is diluted further and applied to agar plates containing the antibiotic. The cells are left to incubate for up to 24 hours and then the number of colonies determined. Calculating the transformation efficiency gives us a method to determine the extent to which the transformation occurred. 3.0 Methods and Materials 3.1 Materials The equipment and materials required for this experiment are outlined in this section. Equipment Required: A shaking incubator operating at 37ÃÅ'Ã…  C A non-shaking incubator An electroporator Materials Required: Cells treated for competency 2 agar plates with ampicillin with a concentration of 100 Â µg/ml pUC-19 plasmids 0.1 cm cuvettes Ice in an ice-box Deionised ultrapure water S.O.C. medium at room temperature 2 tubes with snap caps with a volume of 15 ml 3.2 Sterile Technique Sterile Technique is a must when handling pathogenic strains of bacteria. In this experiment, nonpathogenic bacterial strains are employed. However, using sterile technique is still good experimental procedure and promotes safety. Using sterile technique prevents errors in experimental results by preventing contamination from the surroundings. It also prevents contamination of the surrounding environment by the bacterial strain. Steps employed to prevent contamination included: Carrying out the experiment in an uncluttered area. Utilizing a fume hood to perform all procedures involving the bacteria. Washing hands both before as well as after the experiment Disposing off all bacterial waste in the appropriate container for bio-hazardous materials. 3.3 Procedure 3.3.1 Preparation for Electroporation The 0.1 cm cuvettes were cooled on ice. The electroporator was prepared based on prescribed settings. In order to bring the S.O.C. medium to room temperature, it was removed from the ice box. The cells and plasmids were allowed to thaw in the ice-box. Plates were heated at 37ÃÅ'Ã…  C to prepare for the incubation process. 3.3.2 Procedures I Â µl of pUC19 control DNA and 1 Â µl of ultrapure water were added to 2 separate microcentrifuge tubes with the aid of a pipette. The tube was then placed in the ice-box. 25 Â µl of competent cells were added to each of the microcentrifuge tubes. The contents of the tubes were gently mixed. Care was taken to avoid usage of the pipette for mixing. The tubes were then returned to the ice-box for 1 minute. The contents of each microcentrifuge tube were transferred to a cuvette using a pipette. It was ensured that the cells made contact with the cuvette walls and that no air-bubbles were present. This step was done rapidly to prevent heating up of the cells. The cuvettes were then electroporated. 250 Â µl of S.O.C. medium was added to the cells immediately after electroporation. Each of the two suspensions was transferred to a 15 ml tube. The shaking incubator was then set to 225rpm and used to incubate the cells for an hour to allow expression of the acquired antibiotic resistance. 10 Â µl of the transformed sample was then added to 90 Â µl of S.O.C. medium. The plates containing the ampicillin were then used. 20 Â µl of each of the two diluted samples from step 7 was added to a plate. Even spreading of the sample on the agar medium was ensured. Using the non-shaking incubator, the plates were incubated at 37 ÃÅ'Ã…  C for a day and the results recorded. 4.0 Results and Discussion 4.1 Results Answers to Questions (1) Schematic of observations of the agar plates: Figure 1: Results as Indicated by the Agar Plates (2) Count the colonies and calculate the transformation efficiency. Number of colonies observed = 13 Figure 2: Calculation of Transformation Efficiency Using the formula shown in figure 2, Transformation efficiency = 1.78 1010 transformants/Â µg plasmid DNA 4.2 Discussion Answers to Questions (1) Define the vocabulary used in this experiment: transformation, electroporation, host, plasmid, and competent. -Transformation Transformation is a process of horizontal gene transfer whereby DNA present in the environment of a cell is taken up by the cell. In this experiment the transformation involves the uptake of a plasmid containing a marker that results in ampicillin resistance by E. Coli bacteria through electroporation. -Electroporation Electroporation involves subjecting cells to an electric voltage to create holes in the cell wall. External material can then enter the cell through these holes. Natural processes then cause the hole to close and return the cell to its original state. -Host An organism that harbours a parasite is called a host. -Plasmid A plasmid is circular extra-chromosomal DNA. -Competent A competent cell is one which can internalise DNA present in its external environment. Competence can either be natural or artificial. (2) State why E. coli is used in many genetic engineering experiments. The popularity of Escherichia Coli for genetic experiments is due to various reasons. Firstly, most E. Coli strains are non-pathogenic and pose no harm to humans. Safety is a significant factor in the laboratory and E. Coli use is generally safe. Secondly, E. Coli grow easily and can be duplicated through metagenics. Thirdly, their genetic make-up is relatively simple and can be manipulated with ease. Fourthly they have been extensively studied and a lot is known about them. This makes it easier for researchers and they therefore prefer to use E. Coli for genetic engineering experiments. (3) Explain why competent cells, ampicillin, and S.O.C. medium were used for the transformation. Competent cells are necessary as transformation involves taking external genetic material into the cell. If cells are not competent this cannot happen and the experiment cannot be carried out successfully. Ampicillin is an antibiotic. Specifically, it is a bacteriostatic for E. Coli. It helps distinguish between bacteria that have taken up the plasmid and those that have not. This is because the plasmid contains a marker that causes ampicillin resistance. E. Coli cells do not naturally contain the genetic sequence that causes ampicillin resistance. Thus, ampicillin selection is possible to distinguish between transformed cells and untransformed cells. S.O.C. medium contains the nutrients required to help cells stabilise after electroporation. Electroporation introduces holes into the cell wall of the cell and therefore causes destabilisation of the cell. S.O.C medium contains yeast extract and other nutrient sources that help the cell recover. Once the cell has recovered and if the plasmid has entered the cell during electroporation, the cell will multiply and give rise to a colony during the incubation period. (4) Explain the purpose of the controls in this experiment. The control in this experiment constitutes bacteria without the plasmid that inculcates antibiotic resistance. Without this extra piece of genetic information to enable the bacteria to mount defences against the attack of the antibiotic, ampicillin is this case, the bacterial cells will be unable to multiply in a medium that contains the antibiotic. The cells that were treated such that they could incorporate the plasmid DNA into their genetic make-up will be able to multiply in a medium where ampicillin is present as long as there are enough nutrients available for growth. Thus, the control helps us show that the DNA plasmid was indeed taken up and incorporated into their genetic make-up by the bacteria. The only way for E. coli to have survived with ampicillin present is if they had taken up the plasmid and transmitted it to all generations when they reproduced after uptake of the plasmid. Hence, the control serves to confirm uptake of the plasmid as well as its transmission to fol lowing generations by comparing it to cells in the control that did not have the extra DNA. (5) Explain how the colony growth relates to gene transformation. A colony of bacteria stems from the binary fission of one single bacterial cell. When bacteria reproduce vertical genetic transfer occurs whereby the offspring has the exact copy of the genetic material of the parent. In this experiment, bacteria are introduced into a medium containing the antibiotic ampicillin. E. Coli bacteria with their original genetic make-up will be unable to reproduce due to the presence of the antibiotic as they do not have the means necessary to resist antibiotic attack. This is what is expected in the control sample as ampicillin is a bacteriostatic.. The positive sample on the other hand has bacteria which have undergone horizontal gene transfer by transformation. The plasmid DNA that was used for the transformation process contains genetic code that results in E.Coli developing ampicillin resistance. Thus, bacteria that can incorporate this plasmid and pass it on to their offspring by vertical gene transfer can grow in the environment. This is how colony growth relates to gene transformation. (6) Describe how ionic strength of DNA solution affects electroporation. The ionic strength of DNA solution comes into play due to the electroporation stage where holes are created in the bacterial cell wall to allow uptake of the plasmid by transmission of an electric voltage. For this step, the ionic strength of the solution must be low. If the ionic strength is high, arcing will occur. Arcing is visible during the experiment by sparks and a sound like a micro-scale thunderclap. It can cause cell death as well as equipment damage. Thus, for the experiment to be carried out successfully and to safeguard the apparatus, the DNA solution must be of low ionic strength. (7) If your transformation efficiency is lower than 1 109 cfu/ÃŽ ¼g, conjecture and explain potential reasons for the low efficiency. The transformation efficiency is greater than the benchmark stated above. This corresponds to good transformation efficiency and indicates a successful transformation process. However, the close clustering of the colonies makes it possible that some of the colonies are satellite colonies rather than transformed colonies. The experiment could be repeated with a higher concentration of ampicillin to obtain more reliable results. (8) Discuss current and potential applications of gene transformation techniques in biotechnology. Gene transformation techniques play a crucial role in biotechnology. This is because gene transformation provides a method to produce copies of desired DNA sequences. This is especially useful in the pharmaceutical industry to develop medications that are target specific. Also, this could potentially lead the way to genetic engineering, where defects to the genetic code could be repaired and desired traits inserted through addition of the corresponding DNA sequences. Gene replacement therapy could prove to be the cure for nearly all diseases that take human lives contemporarily. In the future gene transformation could be used to engineer human beings and other animals and plants according to desired specifications. Genetic transformation is also used in the development of pest-resistant crops, which could potentially increase the productivity of the land. This could be key to feed the ever-growing population as the quantity of agricultural land decreases. Understanding the evolution of drug resistance could help us devise ways of preventing drug resistance as well as developing drugs that can overcome resistance. In this arena gene transformation plays an important role horizontal genetic transfer is a natural process in bacteria. 4.3 Sources of Error and Suggestions for Improvement There are a few sources of error that could result in incorrect conclusion being drawn from experimental results. (i) The number of colonies seen need not correspond to the bacteria that transformed. This could be due to the growth of satellite colonies. Large bacterial colonies will secrete beta lactamase, which is what causes ampicillin resistance. Thus, the area around the colony will contain this secretion and be ampicillin-free. A satellite colony could grow in this area from untransformed cells. To avoid this problem, the incubation period should strictly be restricted to 24 hours. Satellite colonies emerge after a delay. By ensuring that results arr recorded promptly, the interference in results brought about by satellite colonies can be minimised. Another method is to use a higher concentration of ampicillin. More time will be required to create a antibiotic-free zone around a colony if the concentration of antibiotic is high. (ii) Identifying the number of colonies can be difficult, especially if the size of the colony is miniscule. This could result in an incorrect calculation of transformation efficiency. In order to increase accuracy of results, a different selection marker can be used. Some selection markers have properties that can be distinguished by shining UV light and other such techniques which result in a high contrast. Using these markers may result in higher reliability of results. (iii) Distinguishing between colonies can be difficult if they grow close to one another and appear to be one large colony. Also, closer colonies would also result in a higher chance of there being satellite colonies. To minimise this problem, crowding on the plate must be minimised. For that, a higher concentration of ampicillin could be used, carbenicillin selection could be used instead of ampicillin selection (although expensive) or the nutrient dilution could be adjusted such that it discourages very rapid proliferation. 5.0 Conclusions The objectives of this experiment were to explore the phenomenon of gene transformation and the development of antibiotic resistance in bacteria as well as to inculcate the practice of sterile technique for handling bacteria. Gene transformation was observed with the development of ampicillin resistance in transformed Escherichia Coli bacteria. The bacteria not exposed to the plasmids containing the genes for antibiotic resistance did not grow in an environment containing the antibiotic while the transformed bacteria formed colonies in the same environment. A calculation of transformation efficiency returned a value of 1.78 1010 transformants/Â µg of plasmid DNA, which is greater than the threshold of 109, indicative of a successful experiment. However, the possibility of some of the 13 colonies of bacteria being satellite colonies as opposed to transformed colonies reduces the reliability of the results. Methods to increase reliability of results were therefore suggested. References 1. Port, Tami. (2008, June 14). Bacteria Horizontal Gene Transfer. suite101.com. Retrieved 3rd April, 2010 from http://bacteriology.suite101.com/article.cfm/bacteria_horizontal_gene_transfer 2. Metzenberg, Stan. (2002). Bacterial Plasmids. California State University Northridge Department of Biology. Retrieved 4th April, 2010 from http://escience.ws/b572/L2/L2.htm

Significant Event

One of these events stands out in particular to me and had he greatest impact in my life. Physically disciplining a child has a dramatic impact on both the child as well as the parent. As a child I remember that for any little misbehaver resulted in a physical punishment as well as getting yelled at, as time went by my mind was set that I already knew when the â€Å"beating† was going to come. My family continue to be very big on respect and discipline, but at the time little did they know that by hitting us they weren't just punishing us for a little while, they were in fact pushing us away and causing frustration, at times even hatred.I also researched that in result of physically harming children increases their risk of mental illness, in which resulted when I came upon this article which states that â€Å"It's pretty well established that physically harming children has a negative impact on mental health, but this is showing the same effect even when you look at milder fo rms of physical force. This is saying that physical punishment should not be used on children of any age. † Each perspective has its strengths and weaknesses, which brings difference to our understanding of the human behavior.In my situation am going to go with the behavioral perspective, as well as the psychodrama perspective. By looking at the physical punishment from a behaviorism perspective, conditioning by pain requires that the consequence always occurs immediately after every incident. The psychodrama perspective states that in childhood certain incidents may occur that produce behaviors in their adulthood. Many different conflicts throughout childhood development shape overall personality. Observational learning refers to learning that occurs as a function of observing, and placating behavior observed in which is particular during childhood.I later figured out that the way my mother and aunts were physically punishing me, resulted in the fact that they learned from my grandmother, they completely mimicked the way my grandmother punished them. As I grew older I began to express myself to them in a matter that I felt curious I began to question why would they always physically harm my cousins as well as myself, their excuse still remains the exact same in which they say † That it is simply something they grew up seeing on a daily basis†. SST memories are not always accurate which can result in a blur, but do believe that it depends on how intense was the situation. There are many memories do vividly remember from my childhood but they are also plenty which don't necessarily remember everything, at times will looking at certain photos and remember exactly what I did that particular day, so I do think that it all just depends on the importance of the situation. Although do in fact accurately remember many different situations in which was hit, many of which I now think back ND know that could have been easily solved with just a simple co nversation.

White King, Red Rubber, Black Death

White King, Red Rubber, Black Death â€Å"White King, Red Rubber, Black Death,† illustrated how King Leopold II of Belgium acquired the Congo as a free state and exploited it by reign of terror. King Leopold II took over leadership of Belgium from his father, hoping to gain power and wealth, as well as assuming control of overseas territories like most other European nations of the time did. Leopold created, through political lobbying and military force, the Belgian Free State.The Belgian Free State gave Leopold power over the African territory of the Congo, which he soon exploited for its large supplies of ivory and rubber. Leopold and his soldiers used the Congolese natives as forced labor, and those who refused to work for the Belgians or who violated their newly established laws were punished by mutilation, torture, or death. Nearly ten million people were either slaughtered or worked to death in the Congo under Leopold's rule.In the end, Leopold's reign of terror in the C ongo became a scandal during the last years of his rule, and he destroyed most of the documents pertaining to the Belgian Free State prior to his death. Before this History class, I would have never had known about the documentary â€Å"White King, Red Rubber, Black Death† and about the many issues that were present in the Congo of Africa in the past. This documentary had left me speechless and astonished in the end. It had truly opened my eyes to the many tragedies that the Africans of the Congo endured under the reign of King Leopold II.Furthermore, I wondered, why throughout the entire reign of King Leopold II didn’t anyone even try bringing him down and making him pay for his cruel punishments depicted upon the Congo popularity? It amazes me that, during the thirty years that Leopold committed acts of violence and cruelty amongst the Congo, Leopold was still worshiped after by the Belgium’s as the man who colonized the Congo and made the Belgium city wealthy . This is a man who depicted the ignorance and greediness of Belgium.In my eyes, everything that Belgium has today, is out of fraud and lies because of the selfish acts Leopold committed. All of this cruelty, because of the exportation of rubber? I couldn’t believe it with my own eyes and ears, the amount of damage Leopold had caused amongst the Congo and its people just because of his obsession with power and wealth. Yet, if this was an issue that was being presented in today’s society of a specific area many would jump to fight and defend this area.King Leopold conducted many extreme measures in the past for no reason, in my opinion. If I was alive during this period and was one of the many people of the Congo who were enduring this cruelty and pain, I would stand up for what I believe in, save my people, and make a change. I do believe that if such person in today’s society, conducted these acts of punishment they would immediately get the death penalty becau se of the well improved justice system we have today, but why not back then?

All Roads Lead to the Ancient Network of Roman Roads

The Romans created a network of roads all across the empire. Initially, they were built to move troops to and from trouble spots. They were also used for speedy communication and ease of pre-motorized travel. Roman roads, specifically  viae, were the veins and arteries of the Roman military system. Through these highways, armies could march across the Empire from the Euphrates to the Atlantic. They say, All roads lead to Rome. The idea probably comes from the so-called Golden Milestone (Milliarium Aureum), a marker in the Roman Forum listing the roads leading throughout the Empire and their distances from the milestone. Appian Way The most famous Roman road is the Appian Way (Via Appia) between Rome and Capua, built by the censor Appius Claudius (later, known as Ap. Claudius Caecus blind) in 312 B.C., site of his descendant Clodius Pulchers murder. A few years before the (virtually) gang warfare that led to Clodius death, the road was the site of the crucifixion of the followers of Spartacus when the combined forces of Crassus and Pompey finally put an end to the slave revolt. Via Flaminia In Northern Italy, the censor Flaminius made arrangements for another road, the Via Flaminia (to Ariminum), in 220 B.C. after the Gallic tribes had submitted to Rome. Roads in the Provinces As Rome expanded, it built many roads in the provinces for military and administrative purposes. The first roads in Asia Minor were built in 129 B.C. when Rome inherited Pergamum. The city of Constantinople  was at one end of the road known as the Egnatian Way (Via Egnatia [á ¼ËœÃŽ ³ÃŽ ½ÃŽ ±Ãâ€žÃŽ ¯ÃŽ ± á ½â€°ÃŽ ´ÃÅ'Ï‚]) The road, built in the second century B.C., went through the provinces of Illyricum, Macedonia, and Thrace, starting at the Adriatic at the city of Dyrrachium. It was constructed by order of Gnaeus Egnatius, proconsul of Macedonia. Roman Road Markings Milestones on the roads give the date of construction. During the Empire, the emperors name was included. Some would have provided a place for water for humans and horses. Their purpose was to show miles, so they might include distance in Roman miles to important places or the end point of the particular road. The roads did not have a foundation layer. Stones were laid directly on topsoil. Where the path was steep, steps were created. There were different paths for vehicles and for pedestrian traffic. Sources Colin M. Wells, Roger Wilson, David H. French, A. Trevor Hodge, Stephen L. Dyson, David F. Graf Roman Empire The Oxford Companion to Archaeology. Brian M. Fagan, ed., Oxford University Press 1996Etruscan and Roman Roads in Southern Etruria, by J. B. Ward Perkins.  The Journal of Roman Studies, Vol. 47, No. 1/2. (1957), pp. 139-143.   A History of Rome to the Death of Caesar, by Walter Wybergh How, Henry Devenish Leigh; Longmans, Green, and Co., 1896.

Jewish Nationalism Of The Ottoman Empire - 1309 Words

Lets begin as most historians do, in the late 19th century. The Ottoman empire ruled over what is now called Palestine. The population there, according to Ottoman records from 1878 was 87% muslim, 10% christian and 3% jewish. Everyone spoke arabic as the daily language and in Jerusalem the religious population was about equal. In the Ottoman Empire, religions were ensured to live peacefully together. The late 19th century was the golden age of nationalism in Europe and no place was crazier than the Habsburg Hungarian Empire. 10 nations in this empire wanted their own state. In this very nationalistic empire lived a jewish generalist named Theodor Herzl who became convinced that the jewish people needed to leave Europe to settle in their own state. The concept of jewish nationalism became to be known as Zionism. It s important to keep in mind that most zionist jews were secular jews. In 1917 the british government issued the Balfour Declaration in hope of gaining support of the jewish people. The Balfour Declaration, promised the â€Å" the establishment in Palestine of a national home for the jewish people†. This was a strong move considering that the Ottoman Empire hasn t lost the war yet. But it turned out the british couldn’t keep their promise when it came to Palestine. Because a year before the Balfour Declaration the british had secretly promised the french that they would divide the arab territories and the brits would keep Palestine. Furthermore in 1915 otherShow MoreRelatedThe Success And Resilience Of The Ottoman Empire Essay1723 Words   |  7 PagesPrior to the arrival of Nationalism and nationalist movements in the 19th century, Ottoman suzerainty over its non-Muslim subjects in the Mediterranean and its European territories (Balkans and Caucasus) rested on religious identification as the cornerstone of society within the Ottoman Empire. 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Etruscan Culture, Architecture, Art, And Religion - 936 Words

1. What were some key aspects of Etruscan culture, architecture, art, and religion? Little is known about Etruscan culture, but from what evidence that has been discovered we know that they were indigenous people of what we today call Italy and Turkey. Some other things we do know is that they inherited some aspects of their culture from the Greeks such as their art, literature, and religion. The Etruscan architecture, being influenced by the Greeks, had temples with stone foundations that were rectangular, on a raised podium, with a peaked roof. The Etruscans designed their temples and tombs in the Doric and Ionic order. The tombs of the Etruscans were decorated with reliefs depicting daily activities and they filled them with riches, weapons, and vases that may have contained food. Etruscan art depicted activities that were normal activities of the daily life of the Etruscan people. They may have decorated the tombs of their daily lives for many reasons. 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History of the Utilization of Musical Instruments Essay Example For Students

History of the Utilization of Musical Instruments Essay For thousands of years we have seen the use of musical instruments in everyday life and also for religion and tradition. To get a better picture we can go as far back as 67,000 years where we have seen the use of the flute used for religious ceremonies. The first recorded use of this dates only back to 37,000 B. C. where they used drums and flutes for ceremonies of marriage. We see that music has been around for thousands of years and we see that they used everything from animal skin and bones, to using resources like wood to create the sounds like a xylophone. A quick glance at the past shows us that music intricacy throughout the eras plays a big part in the world’s culture. For example, we can look at China who used the flute to tell stories about their mythology, but were limited to making the instruments out of bamboo or bones that they could find. According to Inside sciences article titled, â€Å"Exploring the Evolution of Musical Instruments,† it explains the use of lyres and harps in the biblical time of Abraham. Later into the Middle Ages we see that they bring in cymbals, trumpets, flutes, oboes and even more intricate drums. It is also in this time period that we see the emerging of organized music. In the church’s view we see that although music was popular it began to detract from the spirituality of why people were there. About 600 years later we see that people in the Middle East bring into play the use of xylophones and gongs. This shows the musical historians that music still played a big part in the cultures and societies. Then we waltzed into the Renaissance period, a time of beautiful melodies and beautiful symphonies of music. In the early 1400’s we see that people want to know just how many instruments are being sold and how many are being made. Musical Craftsman began to make books and other documents for the instruments that they are making. This is the time period where we see musicians using cowbells for that added touch in their music. Shortly after we see that others are using hunter’s horns in symphonies with a variety of shapes and sizes. We also see the evolution of many other musical instruments’ such as the violin, flutes, trumpets and also drums. Although sales in the 1400’s and 1500’s were not as high as they should be the intricacy of the instruments continued to increase and the sounds became richer in tone and quality. As the richer people began to use these instruments more we see that their popularity increased because of the sounds that they could make and the sheer numbers that they would use at one time. It proves to the world that this was an age of beauty and of music to the generations ahead. Later on, we find that even the poor have instruments, though they may not be the best they still had a great time using them. It is crazy to think that over the course of thirty thousand years the innovations of music would have been this drastic.