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Unformatted text preview: Cellular Building Blocks and Nutrients Carbohydrates, lipids and proteins, along with nucleic acids, are the principle building blocks of life. Most of these building blocks are extracted fro m nutrients acquired by the respective organisms. These organic compounds are the "stuff" of which cells are made. During this laboratory sessio n we will perform various tests commo nly emplo yed to detect the presence (or absence) of three main groups of organic macromo lecules in commo n foods. Object ives:
· Familiarize the students with the pr inciples and procedures for chemical tests used to detect the presence of three different groups of organic macro molecules, viz. carbohydrates, lipids and proteins
· Prepare posit ive (+) and negat ive () control samples to be used for comparison to results acquired fro m analysis of representative food groups
· Use chemical analys is procedures to determine the kinds of organic macro mo lecules contained in samples of "unknown" so lutions supplied by the instructor Materials: Reagent Solut ions Benedict's so lut ion Iodine so lution Biuret solut ion Sudan IV solut ion Organic Co mpounds Glucose control Starch control Lipid (mineral o il) control Protein control Miscellaneous commo n foods "unknown" so lut ions General Supplies Goggles* Pipettes Beaker (400 mL) Test tube rack China marker Timer Hot plate Clean test tubes Test tube ho lder* *Safet y equipment must be used when handling all chemicals and/or hot tubes, solut ions, etc Methods: Part I. Preparation of positive and negative controls. 1. Clear your work area of all unnecessary items (book bags, coats, etc). 2. Obtain and inventory your group's supplies fro m the laboratory cart and set up eight (four pairs) of clean dry test tubes in the tube rack (note: residues fro m other experiments will co mpro mise the qualit y of your results). 3. Label the" positive" and "negat ive" control tubes for each of the FOUR tests listed in the tables below, respectively. Use the vo lumes of reagents listed in the respect ive tables to prepare the control tubes. Be sure to keep the tubes in order in the tube rack, and retain all of the control tubes for comparison wit h the results of the analyses to be performed on commo n foods and "unknown" so lut ions. Record the chro mogenic (color producing) responses for each of the control tubes in the tables provided in the Results sect ion of this laboratory exercise (p. 9). 4. Conduct the fo llowing four chemical assays using a pipette to carefully add the specified vo lumes (number of drops) of reagents into their respect ive tubes (10 drops equals approximately 0.5 mL of so lut ion). Prepare your control tubes FIRST and record their respect ive results, then fo llow the same procedures to test each of the
Page 1 of 4 ver. 1.6 foods and "unknown" samples for carbohydrate (sugar, starch), lipid (fat, oil), and protein. Record your results for your Control Tubes in Table 1 provided in the Results sect ion of this exercise. Benedict's Test: Test for reducing sugars Posit ive Control Tube Negative Control Tube 10 drops Glucose so lut ion 10 drops water 10 drops water 10 drops water 20 drops Benedict's Solut ion 20 drops Benedict's Solut ion Mix the tube contents and HEAT for 5 minutes in hot water. Carefully remo ve the hot tubes using the tube ho lders, allow them to cool, observe and record the results in the table provided. (using a white piece of paper as a background may help bring out subtle color). Light blue = Negative test for reducing sugar Green, yellow or orange = Posit ive test for sugar Iodine Test: Test for starch Posit ive Control Tube Negative Control Tube 10 drops Starch solution 10 drops water 5 drops Iodine so lution 5 drops Iodine so lution Mix the tube contents thoroughly. Hold the tubes against a white background and record any co lor change in the table provided. Light yellowbrown = Negative test for starch Purple, vio let or blueblack ± precipitate = Positive test for starch Sudan IV: Test for lipids (fats, oils) Posit ive Control Tube Negative Control Tube 1 drop Mineral Oil 10 drops water 10 drops water 10 drops water 10 drops Sudan* IV Solution 10 drops Sudan IV Solution *Sudan is a lysochrome co mpound (disso lves in lipids and co lors them). Mix vigorously for 3060 seconds; place tubes in rack and let stand for 5 minutes (do not mo ve or agitate before reading results). Examine the tubes under a dissect ing scope by slanting them and observing the watery surface. Negative test for lipids = the die particles are irregularlyshaped fragments on the surface. Posit ive test = round, spherical globules that may appear orange or red on the watery surface. Biuret Test: Test for Protein Posit ive Control Tube Negative Control Tube 10 drops Gelat in (protein) so lut ion 10 drops water 10 drops Biuret Solut ion 10 drop Biuret solution (Biuret Test for Protein, continued fro m previous page) Mix thoroughly and record color change in the table provided Aqua blue color = Negative test for protein (and/or peptides) Royal blue to purple color = Posit ive test for protein (and/or peptides) Part II. What's in this food? Carbs? Fat? Protein? Testing representative everyday food stuffs. Using the analys is procedures you have perfected (?) above test the househo ld foods provided by the instructor. By necessit y the samples have been made
Page 2 of 4 ver. 1.6 into somewhat unappet izing so lutions; however, the principles and procedures remain the same as for the known control substances. As you examine each sample keep in mind that not many foods are pure carbs, or lipids, or protein, but rather are various mixtures. 1. Obtain sets (of 4) clean dry test tubes to perform each o f the four analyses for organic macromo lecules on each of the food samples provided. Use the Positive and Negative Control tubes you have already prepared as your references for interpreting the results of your tests. 2. To perform analysis o f a given food sample simply subst itute 10 drops of the respective food slurry in place of the 10 drops of posit ive control solut ion. Compare the results of each test to those obtained for the known Posit ive and Negat ive control tubes. 3. Record your results for each food sample in Table 2 provided in the Results sect ion of this exercise (p. 10). Part III. What's in this mystery "solution"? Using the chemical analysis procedures used to prepare the control tubes test each of the numbered "unknown" so lut ion samples for the presence of organic macromo lecule content, respectively. Record the results of your analyses in the Table 3 provided in the Results sect ion of this exercise. 1. Carefully clean and dry the test tubes used for previous analyses. Be sure the tubes are cleaned, rinsed and dried thoroughly since residues from previous tests can adversely affect the results of your subsequent tests. 2. Using the chemical analys is procedures used to prepare the control tubes, subst itute the specified number of drops of the “unknown” solut ion in place of the known control solut ion. Results: Table 1. Results of Posit ive and Negat ive Control Tubes Posit ive Control Tube Benedict's Test for Sugars Iodine Test for Starch Sudan IV Test for Lipids Biuret Test for Protein Negative Control Tube Results (continued): Table 2. Food Sample Results Test Food Benedict's Iodine Page 3 of 4 Sudan IV Biuret ver. 1.6 Table 3. "Unknown" Food Preparations Record "+" for Posit ive or "" for Negative for the results of the tests on each unknown Benedict's Test Iodine Test Sudan IV Test Biuret Test 1 2 3 4 5 Page 4 of 4 ver. 1.6 ...
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- Spring '06