This lab report is about flies.

This lab report is about flies. Format 12pt. font Times New Roman or ArialDouble Space (Except Abstract)Page Numbers but not on title page Title Page Must have a Scientific Name Purpose of this LabTo demonstrate which part of the fly thorax cell homogenate and carries out glycosis and which part carries out respiration You can relate to other source just have to cite it.  I posted the methods we used for the experiment but it have to be reword.  You can look up information about the fly and include it in the report.  I included the graphs and some results. All graphs must be label.                          Student’s Name:_______________________________                                           Date:_______________Lab Report Evaluation MatrixGood                   Average                       Poor                    Not PresentTitle Page (5pts)Descriptive but not wordy (5pts)           _______                   ________             _________         _________ Abstract (20pts)250 words or less (2pts)                           _______               ________                 _________            _________Problem investigatedand hypothesis  (3pts)                              _______               ________                 _________            _________Methods (3pts)                                          _______               ________                 _________            _________Major Results (4pts)                                 _______               ________                 _________            _________Conclusions (4pts)                                     _______               ________                 _________             _________Significance ofFindings (4pts)                                           _______                ________                _________             _________ Introduction (20pts)Sufficient Background infoon organism (2pts)                                   _______                ________                 _________             _________Background info on System (6pts)        _______                 ________                 _________             _________Citations (3pts)                                         _______                ________                  _________            _________Clearly stategoals of project (4pts)                             _______                ________                  _________            _________Hypothesis and predictions (5pts)        _______                ________                  _________            _________ Materials and Methods (15pts)Clearly state methods usedso someone else can repeatthe experiment (5pts)                             _______               ________                   _________             _________Purpose of procedures(Briefly explain why you are doing each step) (4pts)_______               ________                    _________             ________No mundane details and nopronouns (3pts)                                        _______               ________                    _________            ________Passive voice and past tense (3pts)      _______               ________                    _________           _________                                                                                                                                                   Good             Average               Poor              Not PresentResults (20pts)Tables and/or figures present (4pts)      _______        ________         _________       _________Figure or table has proper labels(axis labels with units and error bars) (4pts)_______        ________         _________        _________ Figures and tablessummary headings (4pts)                        _______        ________          _________       _________No interpretationof the results (2pts)                                  _______        ________          _________       _________Reference figures in text (2pts)              _______        ________          _________       _________Briefly summarize main points offigures and/or tables in text (4pts)         _______         ________          _________       _________ Discussion (20pts)  Interpretation of results (why) (8pts)    _______         ________          _________        _________Explanation of the significanceof the results (4pts)                                   _______        ________           _________        _________State if your hypothesiswas rejected or accepted (4pts)             _______        ________           _________        _________Citations (1pts)                                           _______        ________           _________         _________Summarize conclusion in abrief ending statement (3pts)                  _______        ________           _________         _________ Literature Cited (5pts)Listed alphabetically (1pts)                       _______       ________            _________         _________Every citation in text ispresent in literature cited (1pts)              _______       ________            _________         _________Correct format of citations (2pts)            _______       ________            _________          _________ Scientific Journal Present (1pt)                _______       ________            _________          _________                            Biology 155 Lab ReportTitle Page 5 points  Abstract 20 pointsThe abstract should be a concise summary of all aspects of your report: background, system purpose hypothesis, prediction, methods, results, conclusion, and relevance. Should be one paragraph on how to perform lab results. Separate Page summarizeProblem investigatedMethodHypothesisMajor resultsConclusion  Introduction 20 pointsgive sufficient background for the reader to understand the problem being investigateddescribe the problem or concept being investigateddescribe the organism or system being used being investigateddescribe the purpose of the investigationstate any hypotheses or prediction that will be tested Methods 15 points Methods should be described what was done and how it was done in narrative style. Should be in 3rd person past tense Results 20 pointsResult should thoroughly summarize you data. There should be text that should describe your data that you obtained and you interpretation of that data. Include tables or figures. Tables should have descriptive titles and all columns should be labeled. Figures should have descriptive captions. Graphs must have all axes labeled. Any simple direct conclusion you can make about your data can be stated in the results. Save broad conclusion or those that require logical development for your discussion. Discussion 20 pointsThe discussion should be developing the major conclusion of your paper. It should relate your results to the concept or problem that was presented in the introduction. It should state whether hypotheses developed on the introduction or rejected are supported or can be rejected. It should relate your results to other people’s work and to the world beyond your experiment. Literature Cited 5 pointsAll literature used to prepare the paper should be cited in the paper wherever it is relevant.   Trial 1       Tube #1234567Did bleaching occurYesNoNoYesYesNoYesTime took for bleaching (min)18nonenone168none25        Trial 2       Tube #1234567Did bleaching occurYesNoNoYesYesNoYesTime took for bleaching.19nonenone1711none28        Trial 3       Tube #1234567Did bleaching occurYesNoNoYesYesNoYesTime took for bleaching.16nonenone1710none27                             Synopsis — This experiment utilizes the method of differential centrifugation, together withenzyme assays, to separate mitochondria from a homogenate and establish where the key processesof respiration and glycolosis are localized in eukaryotic cells.  Objectives –Learn the basis and use of differential centrifugation for cell fractionation.Learn the use of indicator dyes (in this case methylene blue) in chemical experiments.Deduce the subcellular location of glycolysis and respiration from the results of the experiment.  INTRODUCTIONCell FractionationTwo major types of evidence have provided our present concept of the compartmentation ofintracellular function: (1) inferences from microscopic observation, and (2) evidence from actualphysical separation and biochemical analysis of intracellular constituents. This experiment willprovide evidence for the localization of cellular respiration in mitochondria, and that of glycolysisin the soluble portion of the cytoplasm. The purpose of the experiment is not merely to verify thata given function resides in a particular cell part, but rather to introduce you to an extraordinarilyimportant method of studying living matter and to lead you into some of the mental processesrequired to infer how things work at the subcellular level.Note that in this experiment we will be concerned with the separation of cell organelles.Our separation will not be complete; that is to say, we will not obtain pure mitochondria, but willexploit a rapid procedure which lends itself to easy execution while illustrating more complexprocedures used in the research laboratory. Our procedure yields a rich harvest of mitochondriawhich are functional as judged by the most sensitive biochemical criterion available: the ability tocarry out ATP synthesis coupled to electron transport.We will actually use two methods: homogenization and centrifugation. You are alreadyfamiliar with homogenization from the experiment on the enzyme action. Remember thathomogenization produces a solution of soluble cell constituents and a suspension of insolubleconstituents. The latter include intracellular organelles such as mitochondria. These may beseparated from the other organelles and soluble material by means of centrifugation. In acentrifuge, material is spun on an axis of rotation and thereby subjected to a force (centrifugalforce) directed outward from the rotational axis. This force causes the suspended cell organelles tomove away from the axis of rotation down the length of the centrifuge tube. If the centrifuge runsfast and long enough, the organelles will eventually become sedimented at the bottom of the tube(pellet).The rate of sedimentation varies for different kinds of suspended particles. At a given speedof rotation, heavier and larger particles move faster than lighter, smaller ones. With elaboratecentrifugation procedures, nuclei, which are relatively large and dense, may be separated from“microsomes” (bits and pieces of endoplasmic reticulum with any attached ribosomes).Our choice of experimental material derives from the need for high glycolytic andrespiratory metabolism and the easiest possible separability of mitochondria from the solublefraction. We therefore selected insect flight muscle, specifically that of flesh flies (Sarcophaga).As in many other flies, these have extraordinary contractile performance, causing wingbeatfrequencies of many hundreds to over a thousand cycles per second. This is energized byglycolysis and respiration with glucose as a fuel.Biology 155 Laboratory Supplement: 23The most striking features of flight muscle are (1) the enlarged size of the muscle fibrilswhose contractions are responsible for the performance mentioned and (2) the equally striking giantmitochondria. These features lend flight muscle to our present purposes for two reasons: (1) theactivity of glycolytic and respiratory enzymes is very high, permitting easy detection; and (2)owing to the abundance and large size of the mitochondria, they can be isolated in substantialquantity at relatively low centrifugal forces.Recommended ReadingBaker & Allen: The Study of Biology, 3rd ed., pp. 195-232.Keeton: Biological Science, 3rd ed., pp. 137-138, 163-180.Loewy & Siekevitz: Cell structure and Function, 2nd ed., pp.310-314.Raven & Johnson: Biology 4th ed., pp. 193-205.Biology 155 Laboratory Supplement: 24 PROTOCOLSpecial Preparations Before Coming To LabCarefully study the logic of the experiment (see table) in order to understand why each tubeis necessary and why each component is used. Know the function of the substrates glucose andsuccinate in cellular metabolism.Assay for Glycolysis and RespirationTo understand the assay method, it is crucial to know the meaning of the termsglycolysis and respiration. Glycolysis is best defined as the conversion of glucose into twomolecules of pyruvic acid (pyruvate). This conversion does not consume oxygen, andaccomplishes only a partial chemical degradation of a glucose molecule, thus affording the cellonly a partial utilization of glucose’s potential as a metabolic fuel. In contrast to glycolysis,cell respiration consumes oxygen, and pyruvic acid is completely oxidized to carbon dioxideand water. As you should expect, both glycolysis and cell respiration are accomplishedenzymatically; the former is carried out by the glycolytic enzyme system, and the latter by theenzymes of the Krebs cycle and electron-transport chain. NOTE: The usage of the termrespiration in some texts embraces both glycolysis and the conversion of pyruvate to carbondioxide and water, which requires water. The usage here conforms to that of the scientistswho established this field of biochemistry and is more convenient for a variety of reasons.As a consequence of the points stated in the preceding paragraph, a homogenate whichis glycolyzing but not respiring will not use oxygen. If oxygen is utilized, its concentration insolution will fall. We will use the dye, Methylene Blue to indicate when the oxygen is usedup as a result of respiration. When the concentration of dissolved oxygen is very low, the dyebecomes colorless. This happens as a result of chemical reduction of the dye. Reducingequivalents from metabolic intermediates in the Krebs cycle are passed to NAD (or in the caseof succinate, FAD) and from these nucleotides on via the electron-transport chain. The dyeintercepts this passage and gets reduced. The dye color disappears when it is reduced.Therefore, if we supply glucose to a homogenate in the presence of Methylene Blueand the dye becomes colorless after a period of time, we have evidence for both glycolysis andrespiration. If instead of glucose we supply a substrate in the Krebs cycle derived frompyruvate (we will use succinate for this purpose) and the dye bleaches, we have evidence forrespiration but not glycolysis. If we supply glucose to a homogenate capable only ofglycolysis, the dye would not be reduced and any inference as to the occurrence of glycolysiswould have to be based on other data. Be sure you understand the reasoning behind all thesepoints.The strategy of the experiment is designed to reveal which part of the musclehomogenate carries out glycolysis and which part carries out respiration. The procedurebreaks down into four sections: (1) preparation of the homogenate; (2) fractionation of thehomogenate by centrifugation; (3) biochemical analysis of the enzymatic capabilities of thefractions obtained; and (4) microscope observation of the fractions (optional). Students shouldwork in pairs. Because the preparation of the homogenate is a relatively complicatedprocedure, a flow sheet has been prepared for you (see Figure 1). Read the instructions verycarefully.Biology 155 Laboratory Supplement: 25 Materials:1 stoppered shell vial or small Erlenmeyer flask for flies70 flesh flies, Sarcophaga bullata (ice-cold to anesthetize them)1 wire test-tube rack (small mesh)a few paper towels to cut the flies on1 razor blade1 enamel pan1 thermometer1 grease pencil1 piece of plastic wrap1 large plastic beaker filled with crushed ice7 large glass test tubes to hold reagents, homogenate, and centrifuged fractions7 glass reaction tubes (SMALL size, about 3 inches tall)7 plastic pipettes, 1 ml and controls Procedure: This is the methods that should be included in to lab report PUT INTO YOU OWN WORDS Part A- Preparation of homogenate The instructor will assign one team of two students to prepare the homogenizer. This teamshould obtain a clean homogenizer. Chill it for 5-10 minutes before use in an ice bath. Meanwhile, the instructor will distribute 60 flies among the remaining teams. If the flies arekept in a closed plastic tube on ice, they will be immobilized. Cold causes anesthesia in insects. Using the razor blades, QUICKLY (to avoid warming) cut off the wings, legs, heads andabdomens. Save the thorax of each fly. The thorax is the segment of the body where the wingsand legs were attached. The entire class should do this at the same time, as rapidly as possible.Finally, each team should cut each thorax in half (to facilitate grinding tissue). The thoracesshould be put into the chilled glass homogenizer tube as it sits in ice. Add 15.0 ml of ice-cold homogenizing medium to the homogenizer tube (0.32M mannitolcontaining 0.02M phosphate buffer, pH 7.4). The team designated in Step 1 above should now make the homogenate for the entire class.Run the homogenizer up and down into the mix of medium and thoraces until the mixturebecomes thick (like a milk-shake). During this process keep the homogenizing tube on ice. A different pair of students, assigned in advance, should prepare a filtering device. Thisconsists of a 5 inch diameter circle of cheesecloth, two layers thick, wetted with homogenizingmedium (but not dripping), placed in a short-stemmed glass funnel. The center of thecheesecloth should be pushed down as far as the beginning of the stem. The stem, in turn,should be fitted into a 50 ml graduated cylinder. It helps to put the cylinder in a beaker of ice. Transfer the homogenate to the cheesecloth. It should start to filter through. If it does not,shake the funnel slightly to start filtration. If it still does not, squeeze it through by hand,making sure your hands are clean before doing so. Add an additional 10.0 ml of ice-cold medium to the homogenizing vessel and run thehomogenizer to to suspend any residual tissue debris. Transfer the 10.0 ml to the cheesecloth inthe funnel, using this 10 ml to wash down the material trapped on the cheesecloth. Repeat thewashing of the homogenizer and the cheesecloth with an additional 5.0 ml of ice-cold medium.Finally, with clean hands, squeeze out the cheesecloth bag into the funnel. This filtrationshould provide about 30 ml of homogenate in the cylinder. Most of the material held back inthe cheesecloth includes pieces of thoracic integument and large muscle fibers.Biology 155 Laboratory Supplement: 26 Mix the filtered homogenate thoroughly. This is crucial. Measure its total volume, and recordthis value to the nearest mililiter. Transfer 15.0 ml of the homogenate to a clean tube marked H(for whole homogenate). Keep this tube on ice at all times. Transfer the remaining 15 ml homogenate to a clean centrifuge tube and place the tube in abeaker of crushed ice. Prepare a balance tube by putting 15 ml distilled water into a new centrifuge tube like the oneused in the previous step. Place both tubes in the refrigerated centrifuge, on opposite sides of the rotor. Centrifuge at5000 rpm for 20 minutes. Another crucial step: immediately after the centrifuge stops, retrieve the tube containing thehomogenate, carefully holding it at the same angle at which it lay in the centrifuge. Pour all thesupernatant into a clean (rinsed with distilled water and shaken dry) 25 ml graduated cylinder.Do not pour out any of the pellet (the pellet is whitish in color); the point is to achieve a“clean” separation. Restore the volume of the supernatant to 15 ml with the homogenizingmedium; shake well to mix contents. Transfer to a clean tube, mark it S, and keep it on ice.The volume of S must exactly equal that in step 10 above. Add ice-cold homogenizing medium to the pellet. The amount of medium added should be justenough to make the final volume of resuspended pellet exactly equal to the original volumecentrifuged (15.0 ml). This is crucial. Stopper the tube and shake it to resuspend the pelletthoroughly. When the pellet is resuspend, label it P. You should now have three labled tubes on ice: uncentrifuged homogenate (H), resuspendedpellet (P), and supernatant (S). The pellet contains nuclei, glycogen (polysaccharide) granules,mitochondria, and bits of the muscle’s contractile apparatus. The supernatant contains mostsoluble muscle constituents, including glycolytic enzymes and some membranous material(reticulum) which is too small to centrifuge out at the speeds used. Each individual team of students should now obtain 1 ml of H, l ml of S, and 1 ml of P inlabeled tubes. Keep them in a beaker of crushed ice.Biology 155 Laboratory Supplement: 27 FLOW CHARTCut 100 flies, ON ICEDistribute 15 flies/team for preparation of thoracesStudents return halved thoraces (wings, legs, head, and abdomen removed)Grind Pool thoraces in homogenizer, ON ICEAdd 15.0 ml homogenizing bufferHomogenize for 2 minutes, rheostat set at 50Filter Through cheesecloth, into a 50 ml graduated cylinderWash & Rinse homogenizer with 10 ml homogenizing buffer; pour throughcheesclothRepeat Rinse with another 5 ml of homogenizing buffer; squeeze outcheeseclothDivide Pour off 15 ml of homogenate into a labeled tubeCentrifuge The remaining 15.0 ml homogenate for 20 minutes at 5000 rpm.Separate Supernatant (S): Restore to 15.0 ml with homogenizing buffer; mix wellfrom Pellet (P):Restore to 15.0 ml with homogenizing buffer; mix wellDistribute 1 ml of H/team, 1 ml of P/team and 1 ml of S/team  Part B- Biochemical analysis of fractionated homogenateEach team of two students should obtain the following materials in the quantities indicated.Keep them in clean, dry, labeled test tubes in a rack at room temperature, not on ice.Substance Concentration QuantityMannitol buffer* 0.32M 3mlBuffer-cofactor-dye mixture ** 5mlGlucose 0.015M 3mlSuccinate 0.2M 3ml* Mannitol buffer = homogenizing medium** Potassium phosphate buffer, pH 7.4 (0.2M); ATP (0.0125M); MgCl2(0.005M); Methylene blue (0.5mg/ml).Obtain 1 ml plastic pipettes for these solutions, and three pipettes for dealing with tubes H, S,and P. Each pipette should be marked to avoid cross-contamination of solutions and should beleft in the tube rather than placed on the table top.Obtain seven clean, small glass test tubes to be used as reaction vessels in the next step. Besure these test tubes are identical in size. Number the tubes clearly 1-7, using a grease pencil.Obtain a pan containing water adjusted to 35°C, about two inches deep.Biology 155 Laboratory Supplement: 28The following table shows what to add to each of the seven reaction tubes. You will workfaster if you add the first ingredient (mannitol) to all the required tubes, then the buffer mix, andso on down the line. Do not add homogenate, pellet, or supernatant until last, and only whenyou are really ready for the reactions to start. The exact time of adding H, P, and S should berecorded carefully. Be sure tubes H, P, and S are thoroughly agitated before pipetting fromthem) their contents may settle). All volumes shown in the following table are in milliliters.Ingredient Reaction Tube Number1 2 3 4 5 6 7Mannitol 0.25 0.25 0.25 – 0.25 0.25 0.45Buffer mix 0.45 0.45 0.45 0.45 0.45 0.45 0.45Glucose 0.20 0.20 0.20 0.20 – – –Succinate – – – – 0.20 0.20 –Whole Homogenate (H) 0.25 – – – – – 0.25Pellet (P) – 0.25 – 0.25 0.25 – –Supernatant (S) – – 0.25 0.25 – 0.25 –Immediately after adding H, P, or S, each tube should be rapidly and thoroughly mixed. Gentlesloshing is no good. The best index of complete mixing is uniformly distributed dye color(blue) in every tube. Immediately go on to step 6.Place the rack of reaction tubes in the pan of water at 35 °C, noting the exact time. Do notagitate the tubes at all from this point on. The water must be deep enough to come to a levelabove the reaction mixture in the seven tubes. During the course of the experiment it will benecessary to add hot water to maintain the temperature at 35 °C. When you do this, add it awayfrom the tube rack and mix the water in the pan thoroughly without any disturbance, evenslight, to the position of the tubes.As noted earlier, the indicator of reaction in the tube is dye color. When oxygen is exhausted,methylene blue turns from bright blue to colorless. The tube then takes on whatever color itsother contents have, in this case homogenate color. Note the exact time at which individualtubes lose blue color and record this. The time is not necessarily the same for all tubes andsome may “never” bleach, i.e., in more than one hour. Note: the bleaching of a tube dependson metabolism using oxygen faster than it can diffuse back in from the air. When a tubebleaches, you will still see a bluish upper margin at the surface in contact with air. This can bedisregarded for present purposes. It may take 10 minutes or longer before tubes bleach.(Optional for the curious: when a tube has bleached, slight agitation will re-aerate it and turn itblue again. This can be demonstrated easily and the tube will bleach all over again. Why doesit bleach? Why is the time different from step 7?Tube 7 deserves special attention. Among cell biologists, this is loosely termed a “no-substratecontrol tube”. This tube was set up to test a specific question, namely: does the action in tube 1depend on the addition of glucose? What happened to the dye in Tube 7, if anything? Whatconclusions does this permit you to draw? Is there any way in which the results which this tubeprovides can still be consistent with the idea that glucose is the initial substrate for glycolysisand respiration in vitro and in vivo?Biology 155 Laboratory Supplement: 29