Simulated Blood Lab Report

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Simulated Blood Lab Report

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Blood Typing Demonstration Using Simulated Blood

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In order to acclimatize the Daphnia to laboratory conditions, they were then placed onto a petri dish on the Daphnia cooling chamber. The cooling chamber was located on the stereomicroscope platform and brought down the heart rate of the Daphnia to a range that was countable by the observer, since Daphnia heart rate at room temperature is too rapid. On the cooling chamber there were two petri dishes: one for the Daphnia that were going to be tested, and one with the Daphnia being tested on, to ensure constant consistent temperatures for each trial. To maintain a temperature conducive to the heart. Now that testing has been done the providers know what type of medication would be best to start with.

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Examples of enzymes used in this kind of analysis are hexokinase and glucose oxidase. The second method involves the reaction of a type of molecules called amines, molecules that contain an -NH 2 group attached to a carbon. Certain amines will react with glucose to create a colored product. Just as in the first method, the more colored product that is produced, the more glucose that must be present in the sample. Cholesterol is a required component of all cell membranes and forms the basis of many hormones.

Chemically it is classified as an alcohol, having an -OH group attached to a carbon. It is not soluble in water and therefore must be carried through the bloodstream by proteins. Cholesterol acts just like any other solute in a solution, if you put too much in, it will begin to precipitate out. When a high concentration of cholesterol in the bloodstream is saturated at high level, this precipitation results in the depositing of plaques containing cholesterol on the inner surfaces of blood vessels. Continued accumulation of these plaques can cause the passageway to become restricted or even blocked altogether. Atherosclerosis is a disease characterized by hardening of the arteries from the deposits of cholesterol plaques.

This condition can lead to a myocardial infarction heart attack , stroke, or other decreased organ efficiencies. You get cholesterol not only from your diet animal byproducts but your liver which produces grams a day. Cholesterol is carried away from the liver to the other cells of the body by macromolecules called low density lipoproteins LDL. Free cholesterol is removed from the blood and returned to the liver by macromolecules called high density lipoproteins HDL.

Analysis of cholesterol levels in blood serum is similar to that of glucose. It can be achieved through an enzymatic process whereby enzymes react with free cholesterol to produce a product that absorbs light at specific frequencies. This in turn can be detected with a spectrophotometer. The second way involves the reaction of cholesterol with a strong acid. The product is colored and can also be quantified with a spectrophotometer. Proteins are made from one or more long chains of molecules called amino acids. Amino acids are a class of molecules that have a similar structure. Each amino acid also has some other group attached to the central carbon atom, but it varies from one amino acid to another.

Proteins serve a wide variety of purposes in the body. Altogether there are some proteins found in blood plasma. Ten to twelve of the 20 amino acids needed by the body can be produced by the body itself. The rest must be obtained from dietary sources. Control of serum levels of amino acids is primarily controlled by the kidneys, in which amino acids are filtered out of the blood and then reabsorbed.

Analysis of blood serum for total protein can be done with either refractometry or biuret reagent. When light passes from one medium, such as air, to another medium, such as water, it bends. This is called refraction. The bending is due to a difference in density. The addition of a solute to a solution changes its density. Proteins are by far the most abundant substance in blood plasma, so any change in the refractive index is going to be an indication of protein concentration.

In the second method, biuret reagent is used. This involves Cu II ions reacting with particular peptide bonds found in proteins to form complexes of a different color red than the Cu II ions by themselves blue. The detection of this color change can be accomplished with a spectrophotometer. The set of activities that follows was designed to be implemented only after several other concepts have preceded it. Therefore, they will have some acquaintance with standard lab techniques and developing procedures for experiments. In addition, writing and understanding basic chemical equations including acid-base reactions will have been covered just prior to the onset of this module.

This instructor is under the impression safety is something that must be taught, demonstrated, and reinforced at all opportunities. Prior to this module students will have conducted numerous labs where safety rules were thoroughly scrutinized. Due to the use of chemicals in this module, safety goggles should be worn. All containers, cuvettes, etc. Should be labeled. Spillage should be reported immediately to the instructor. Skin exposure should be washed with mild detergent and water.

Eye exposure should be rinsed for 15 minutes and further first aid taken as necessary. Introduce students to three basic chemical reactions used to determine glucose, cholesterol, and total protein in blood serum. Students will balance each reaction. Explanation of how an indicator dye can be used to determine the progress of a chemical reaction. Concentration can also be reviewed. The enzymatic processes of detection and protein-dye complexes will be discussed. Students and teacher as facilitator determine procedures required to test for various concentrations of glucose. Students run first sample concentrations of glucose, collecting data to compile a calibration curve.

Graphs, utilizing a computerized spreadsheet such as Excel are discussed and demonstrated in class. Samples of data are used on the overhead to demonstrate regressional analysis, correlation R 2 and its significance. Students run samples of glucose to determine the effects of temperature on the oxidase and peroxidase reactions. Students run samples of glucose to focus on initial rates of reaction and endpoint analysis; students are focusing on an appropriate length of time for the reaction to proceed.

Students run samples of glucose to determine the effect of pH on oxidase and peroxidase reactions. Follow up questions are answered and conclusions drawn regarding the factors influencing the glucose oxidase-peroxidase reactions. The exact conditions of the tests temperature, pH, water type, endpoint will be determined by students based upon their previous tests. Students conduct research to determine causes that would likely produce the conditions they found.

Students critique several hypothetically engineered designs of instruments for doing quantitative analysis of blood chemistry. This page of the module is designed as a student handout. The glucose oxidase reaction and peroxidase reactions are shown. Students will balance the equation and there will be some discussion regarding an interpretation of the equations. Students will need a review of the wavelength nature of light and a basic understanding of how a colorimeter works. To demonstrate the colorimeter several samples of known concentration can be made up ahead of time.

These samples can then be analyzed by the colorimeter to show higher concentrations of a dye will absorb more light and this is in turn is measured by the instrument. The sets of labs in this module require a fair amount of accuracy as very small changes in concentrations are involved. Past experience has shown this to not always be simple for the junior high student. Therefore, it is important to stress patience, attentiveness to tasks, and accuracy to the students. Standard experimental procedures need to be developed for the class. The question posed is: How do different concentrations of glucose affect the reaction of the glucose oxidase reaction above? Students should come up with responses that show different concentrations of glucose will need to be measured out, reacted with reagent, and then tested with the colorimeter.

Students may suggest other ideas such as: using different amounts of reagent, allowing the glucose-reagent mixture to react for various lengths of time, or running the reaction at several different temperatures. These ideas are student-centered and should be encouraged, for the purpose of the discussion is to have the students think through the development of the procedures as much as possible rather than having the procedures written for them. Students will test out these variables in the following days of the module. In their minds they will be acting on THEIR ideas and this will tend to enhance their enthusiasm in the project. After providing the students with normal human serum glucose concentration levels, they should decide on a range that should be analyzed.

The length of time and amount of reagent to add will not be readily apparent to the student so the data collected at this institute will serve as the initial protocols for these two variables Appendices G,H. Protocols developed at this institute for establishing a glucose calibration curve are below. Teachers may choose to use them directly or have students develop them in the classroom with guided assistance from the instructor.

Procedures: Set up 11 cuvettes in a rack and label them 1 - Add 2. Let cuvettes stand for 15 minutes. Take cuvettes to the Spec 20 one or more stations may be set depending upon accessibility of equipment. Place cuvette 1 in the Spec 20 and calibrate to 0 absorbance. This is the blank all other samples will be compared to. See Appendix K for further instructions on using the Spec 20 spectrophotometer.

Place cuvettes in the Spec 20 one at a time and record absorptance readings in the data table for each cuvette. There is opportunity here to allow the students to practice math skills. Because ordinary blood serum contains glucose concentrations far in excess of what can be analyzed by this method, the samples must be diluted. Students will need to compute concentrations. The math is fairly straight forward, however a review may be necessary. The initial concentration of a solution, after being diluted, is related to its final concentration by the following formula:. Or, rearranging the terms and solving for M final , you get:. The author is of the opinion of showing students how to rearrange the terms rather than just providing the form of the equation necessary for the students, even though some students at this level may not yet have had beginning algebra.

Each lab group now has a set of data which includes an absorbance reading for varying concentrations of glucose. It may be necessary to assume that students are unfamiliar with using Microsoft Excel so a demonstration will be required. Using data from this institute, students will be shown how to set it up in the spreadsheet and then how to make the graph using Excel see appendix L. A projection unit which can be directly connected to the computer or an overhead unit makes this demonstration very feasible in the classroom. Various amounts of information on how to manipulate the charts e. The purpose of collecting this data was to see if a calibration curve which should be linear could be achieved. Students will be shown how to do this on Excel.

If the data is so scattered and its correlation to the linear best-fit line is relatively low, then accurate deductions from its use in determining unknown concentrations will be unreliable. Other items related to interpretations and significance of data should be discussed. Therefore a regression analysis is done to show whether or not a strong correlation is present in the data.

After the demonstration to the class, students should be able to sit down at a computer and graph their data, plot a trendline, determine its slope, and determine the correlation among their data points. As part of that class discussion, many variables that would affect the rate of reaction should have been uncovered as mentioned previously page Students will be able to discover for themselves if any of these variables may have on the outcome of their data. One of these factors that may affect the reaction rates of the glucose oxidase and peroxidase is temperature under which the reaction takes place. It is not necessary for the students to prepare all of the sample concentrations used in the Activity 2 for a couple of reasons; one, we may assume if temperature has an effect on the reaction rate on one concentration it will have a similar effect on all other concentrations, and second, for the purpose of reducing the necessary amounts of reagents required for these activities, each group can test the effects at one or two temperatures and the data compiled together as a class.

Each group is assigned a specific temperature range e. Groups will make their respective water baths by mixing varying proportions of hot and cold water from the aforementioned containers. The following chart illustrates the range of temperature baths that can be established in this way: Group number volume cold water volume hot water estimated temp. Vortex the cuvette and place in the water bath. Allow to stand 5 minutes. Record the temperature of the bath at this point. Remove the cuvette, take it to the Spec 20, and determine its absorbance. Students can then fill in their own data charts once the class data is completely filled in on the overhead. In this activity students will investigate the procession of the glucose oxidase-peroxidase reaction as a function of time.

Students will collect data showing chemical reactions do not always proceed linearly i. The reason for this is that the concentrations of the reactants and products are changing with time. If needed, students can be encouraged to think along the lines of what is changing in the cuvette over the period of time as the reaction is taking place. This activity requires the use of a colorimeter and CBL TM system calculator and software to link the two instruments together. Directions for its use can be found in Appendix M. The following activity is designed with each lab group using the same concentration of glucose.

In reality, chemical reaction rates depend on the initial concentrations. Therefore, a variation of this activity would be to assign each group a different concentration and to compare the data among the class groups. Procedures: Obtain 2 cuvettes and label them Place 1. Add 10 drops glucose solution to cuvette 2. When ready, add 2. After the sample has run for the desired length of time 15 minutes remove the cuvette, collect data, and graph. What happens when the concentration of hydrogen ions is low? Or high? How will this effect the ability of glucose to react with glucose oxidase? It is this question that is the focus of this experiment. The glucose solution prepared for the other activities in this module has had a pH buffer added to it.

The term pH refers to the concentration of hydrogen ions present in a solution. The purpose of a buffer is quite literally, to maintain the pH of a solution. A buffer is a solution made up of a weak acid and its conjugate base in equilibrium such that their concentrations are far in excess of the hydrogen ion also present in the solution. Buffers are able to maintain a fairly constant pH by two ways: 1] if an acid is added to the solution, the conjugate base of the buffer reacts with the free hydrogen ions of the added acid to maintain a fairly constant concentration, and 2] if a base is added to the solution, thereby reacting with the free hydrogen ions, the equilibrium of the acid-base of the buffer is driven more toward the base thereby providing more free hydrogen ions and once again the concentration stays relatively constant.

Students will need some understanding of acids and bases to appreciate the importance of this experiment. If acids and bases are not covered prior to this module, it is suggested the instructor take a couple of class periods briefly to review the concept and then expand students familiarity with the subject by doing one or more simple qualitative titration labs. Procedures: Obtain 4 cuvettes and label Obtain dropping bottles one containing glucose solution, one containing glucose reagent, one containing. Measure out 1.

Place this back in the test tube rack, it will be used as the blank for the spectrophotometer. Measure out 2. Use the table below. After adding drops, vortex the cuvette and check the pH with the pH probe. Continue to repeat the process until the desired pH is achieved. This will provide a glucose concentration of 2. Vortex the cuvettes and allow to incubate for 15 minutes. Take the cuvettes to the Spec 20, zero the Spec 20 with cuvette 1 and record the absorbance of each of the other cuvettes.

Students will write a conclusion for each of the experimental activities conducted so far 2,4,5,6. The author is under the belief students should work collaboratively together in their lab groups while doing this activity. Various methods could be utilized if needed for individual assessment. Each conclusion should consist of the following: Description of what occurred when the experiment was conducted. Student responses should be descriptive, noting errors made by the group that may influence the results, the appearance of the various cuvettes throughout the reaction process, trends in the data, and exceptions to these trends.

Explanation of the pattern s found in the data or lack thereof. Explanations should demonstrate that the student is compiling concepts from throughout the overall course. For example, an increased absorbance with temperature would indicate more glucose being oxidized, for at increasing temperatures the number of molecular collisions would be increased per unit of time due to the faster movement of the molecules. In addition, students should address the questions on the following page in writing their conclusions.

Activity 2 How does the absorption of light depend on the concentration of glucose? Are readings of much more than 1. What can be done with the solution from above so that the absorption reading is between 0 and 1? Activity 4 How does temperature affect the amount of glucose that is oxidized by the reagent? What range of temperatures is important to look at? Where was the rate the fastest? What is the minimum amount of time required for the solution to reach its highest absorbance reading?

If this test was done in a clinical setting, would you always have to let the reaction run for this period of time? Why or why not? Activity 6 What is normal human serum pH? At what pH did the reaction proceed the farthest? Students will now find a calibration curve for cholesterol concentrations, similar to the process used to determine a glucose calibration. It will enable them to determine the concentration of cholesterol in an unknown sample. After providing the class with some information regarding normal human serum levels of cholesterol, students should identify these levels as being similar in order of magnitude to glucose levels.

Having run previous samples of varying concentrations of glucose and determining their absorbance values, thus gaining some knowledge as to the range of concentrations that will work, students should come to the conclusion that a similar range of concentrations will need to be established for a calibration curve of cholesterol. In fact, the same range of concentrations will work quite well dilution. Once again, the protocols below were established and tested during the institute and may be used as is or students can be given the opportunity to develop their own set of procedures.

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