Physiology 12 Lab, M. J. Malachowski, Ph.D
- Pancreatin is a commercially prepared mixture of all the pancreatic enzymes. The protein to be used was obtained by adding a small amount of egg albumin to a large volume of hot water, resulting in a flocculent precipitate of coagulated egg white. A beaker containing the precipitate and a small amount of water will be distributed to each team of students.
Number a series of clean test tubes from 1 to 3 and place 2 ml of the precipitated egg white (not the fluid part) into each tube. Add the following substances to these tubes:
Place all three tubes into a water bath at 37 oC for 1 1/2 - 2 hours. Note any change in their appearance at the end of the laboratory session.
- Add 5 ml of milk and 3 ml of 1% litmus solution to each of 4 test tubes. To tubes # 1 and # 4, add 0.5 ml of distilled water and to tubes # 2 and # 3, add 0.5 ml of 2 % pancreatin solution. Add a pinch of bile salts to tubes # 1 and # 2. Tube # 4 will be used as a standard for color comparison. Place all the tubes into the water bath at body temperature for 30 minutes. Litmus solution is used as an indicator in this experiment. The end point of fat digestion is indicated by a color change to violet or red. Compare tubes # 1, # 2, and # 3 with # 4 and note any difference in color Compare tubes # 2 and # 3 more closely.
To each of 2 tubes, add 3 ml of corn oil. To one of these add 3 ml of distilled water. Shake vigorously. To the second tube, add 3 ml of distilled water and a pinch of bile salts. Shake vigorously. Compare the appearance of the two tubes.
Place 5 ml of 1 % starch solution in a test tube and warm the tube in the water bath (37oC) for about 10 minutes. Label this tube Pancreatic Starch Digest (PSD). Add a drop of Lugol's solution to each depression in a depression test plate; note the color. Using a Pasteur pipette, add 1 drop of the PSD starch solution to the Lugol's solution in the first depression on the test plate; note the color. Add 0.5 ml of 2 % amylase to the PSD starch tube and mix well. At 30 second intervals, test the PSD solution for starch by adding two drops to the Lugol's solution in the next and then the next, following, depressions in the plate. Note the amount of time it took for the solution from the PSD tube to stop causing a color change when it was added to the Lugol's solution. (Add up the 30 second increments.) When the PSD solution stops eliciting a color chang upon addition to the Lugol's solution, test the remaining solution in the PSD tube for sugar (What sugar is this? Note this.) with Benedict's solution. This sugar test is done by adding 5 ml of Benedict's solution to the tube and placing the tube in a boiling water bath. Observe any changes in color in the tube after 10 minutes.
Diatase is an enzyme occurring in plants. It has the same action as salivary amylase, converting starch to maltose (a disaccharide). To each of 4 test tubes, add 5 ml of 1 % starch. Before adding the diatase, place each of the 4 tubes into the following water baths for 5 minutes.
After the tubes have been in the baths for 5 minutes, add 10 drops of 1 % diatase to each tube. Note the time at which the diatase was added to the tubes and allow the tubes to remain in their water baths for exactly 5 minutes following addition of the diatase. Next, place all 4 tubes, at the same time, into a boiling water bath and add 5 ml of Benedict's solution to each tube. Observe the change in color of each tube.