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Flow Chart - Labor Laboratory atory # 1: Digestion – Enzymes- P Part art 1: P Pepsin epsin Dig Digestion estion of Albumin Do you think that you should be performing another control(s) other than tube 4?

Cut albumin (the white of a hard-boiled egg) into 1 4 uniform cubes of about 1-2 mm on each side

Place two cubes of albumin into each of 8 test tube

Label tubes 1.....8 and fill tubes as follows

1. 3 ml 5 % Pepsin solution 2. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution (DO NOT use 0.1M HCl) 3. 3 ml 5 % Pepsin solution + 5 drops 1M NaOH solution 4. 3 ml H2O (this is a control for no pepsin) 5. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution 6. 3 ml 5 % Pepsin solution + 5 drops 1M NaOH solution 7. 3 ml 5 % Pepsin solution (preboiled for 10 min) + 5 drops 1M HCl solution 8. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution + 1 dosage of antacid*

Gently swirl to mix solutions. Don’t Spill.

Antacid preparation – to a beaker, add 20 ml 0.1 M HCl (determine pH). To this solution add one dose of antacid. If required, use a mortar and pestle to crush tablets. Add 3 ml of this solution to test tube 8

Tubes 2, 5 and 7 should be very acidic (pH = 2ish) and tubes 3 and 6 should be basic

Measure the pH with pH paper.

Incubate test tubes 1-4 and 78 for 90 min at 37°C

Incubate test tubes 5 & 6 for 90 min at room temperature (about 20°C).

Shake the rack gently from time to time to keep the contents evenly mixed

After the samples incubate for 90 min

Measure the pH of each tube

Create a qualitative scale to determine the amount of protein digestion.

Flow Chart - Labor Laboratory atory # 1: Digestion – Enzymes- P Part art 2: Digestion of Starc Starch h by Amylase Obtain 15ml of prepared amylase (0.1% w/v) in a small beaker

what controls might you want to perform? When you have determined the controls (consult with yourTA)

Label tubes A, B, C, D, E, F and G add:

A. 2 ml amylase + 5 drops 1M HCl B. 2 ml amylase C. 2 ml water D. 2 ml amylase E. 2 ml water F. 2 ml amylase G. 2 ml water

Incubate A, B and C at 37°C (body temperature) for 90 min

Examine the physical characteristics of your amylas

i.e. color, viscosity, pH, turbidity

Add 2 ml of starch solution (2% w/v) to each of the test tubes mentioned above for a total volume of ~4ml.

incubate D and E for 90 minutes at 20°C (room temperature)

Incubate F and G on ice.

After incubation divide samples into two sets of test tubes [mark A1 - G1 and A2 - G2]

Lugol’s test for starch: Put one drop of lugol’s solution into each of tubes A1 - G1. A dark blue/purple color indicates the presence of starch, shades of light grey indicate lesser amounts of starch.

•Place data in a table that is clear and concise and describes the results of the experiments performed. Remember to include a detailed legend. •Describe the conditions necessary for digestion of starch by amylase.

Benedict’s test for sugar: Add 2 ml of Benedict's solution to each tube (A2 – G2) and place tube in boiling water for 2-5 minutes. Read Benedict's test as follows:

Colour Interpretation* Blue No sugar Green Slight sugar Yellow Some sugar Orange Lots of sugar Red All sugar * (Precipitate indicates sugar)

Flow Chart - Labor Laboratory atory # 1: Digestion – Enzymes- P Part art 3: Digestion and Emulsification of Lipids Observation of emulsification of fat by bile salts

Place two clean test tubes in a test tube rack

Add 1 ml vegetable oil to each tube

Observation of lipase activity:

Pre-incubate the litmus cream and 2% pancreatin solution at 37°C for 5 minutes.

Place 4 clean test tubes in a test tube rack and add to each:

Add 2 ml distilled water to each tube

Add a tiny amount of bile salts to tube A

Cover each tube tightly with a square of parafilm and shake vigorously for about 1 minute. Do you notice any difference between the two tubes? Observe for 10-15 minutes and record your observations.

A. 2 ml cream and 2 ml 2% pancreatin B. 2 ml cream and 2 ml distilled water C. 2 ml cream and 2 ml 2% pancreatin and small pinch of bile salts D. 2 ml cream and 2 ml distilled water and small pinch of bile salts

Cover each tube tightly with a square of parafilm and shake to mix solutions

Measure and record the pH of each tubes contents. Do you think the pH of the solution would affect the activity of pancreatin and why? Can it be tested under these experimental conditions?

Test for lipase activity: We will observe the activity of pancreatic lipase by adding lipase to a litmus cream preparation. As the lipase hydrolyzes triglycerides in the cream to fatty acids, the hydrogen ions from the fatty acids should lower the pH and change the color of the litmus indicator from blue (negative for lipase activity) to red (positive for lipase activity).

Incubate each tube at 37°C for up to 1 hour. Observe the changes (each 10 min.), if any,in the color of the samples. Keep track of the time of any changes.

At the end of the incubation, test the pH and record in a table.

Note the odour of each tube.

Flow Chart - Labor Laboratory atory # 1: Digestion – Enzymes- P Part art 4: Mecha Mechanisms nisms of Food Propulsion and Mixi Mixing ng

This part will be performed outside of the lab in the hallway. Eating and drinking is not allowed in the lab. Remove your lab coat, goggles and gloves before exiting the lab and wash your hands.

Obtain a cup of water, a stethoscope, and an alcohol swab.

Swallow a mouthful of water consciously note the movement of your tongue during the process.

Repeat step 2; lab partner should watch the externally visible movements of your larynx

Lab partner should clean the earpieces of the stethoscope with an alcohol swab and place in their ears.

Place the diaphragm of the stethoscope over partners abdominal wall (or your partner can do this themselves) approx. 2.5 cm below the xiphoid process and slightly to the left, to listen for sounds. Repeat step two

Determine the time interval between these two sounds. This gives a fair indication of the time it takes for the peristaltic wave to travel down the 25 cm of the esophagus.

Note: you should hear two sounds – one when water splashes against the gastroesophageal sphincter and the second when the peristaltic wave of the esophagus arrives at the sphincter and the sphincter opens, allowing the water to gurgle into the stomach....