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Biofuels Webquest: Spitting


Did you know that there is an enzyme, in your saliva?


Amylase, is not only an enzyme in your saliva, but it's the enzyme chosen for study in this lesson.  So what's up with enzymes in your mouth?  Amylase begins the digestion of starch in your mouth.  Starches are foods like breads, potatoes, pasta and rice.  Starch is made up of many linked glucose molecules. Amylase breaks this starch into glucose, which is easily absorbed into your blood and used in your cells for energy. 

Would you like to see what happens to food when it's eaten? Here is a video of mouth parts and digestion:




To observe the reaction between iodine and starch, add one drop of starch solution to one drop of iodine  in the first well of a spot dish. Describe the reaction.


Since the enzyme digests starch, this color change will not occur if the starch has been completely digested. If the starch has only been partially digested, the color will be reddish, or intermediate between blue-black and the amber color of the iodine. 


The appearance of the blue- black color is a positive test for the presence of starch. If there is no blackish tinge, starch is not present.

You tube video Background on the Amylase lab part 1


You tube video Background on the Amylase lab part 2


Chemical reactions require some sort of activation energy, in much the same way as your car needs a fully charged battery to start.  A catalyst is a chemical structure that lowers the activation energy needed to start a reaction. Enzymes are a particular kind of catalyst - they are proteins which frequently participate in only a limited range of specific reactions. The enzyme combines with the reactants, forming an enzyme-reactant complex, which then separates, leaving the product of the reaction and the unaltered enzyme.

Amylase, a component of your saliva and also produced by your pancreas, is the enzyme chosen for study in this experiment. It begins the digestion of starch in your mouth or small intestine. Starch is made up of many linked glucose molecules. Amylase breaks this starch into glucose, which is easily absorbed into your blood and used in your cells.  The action of amylase can be easily observed using the iodine test.  Starch mixed with iodine produces a deep blue or blue-black color. Maltose leaves no color when mixed with iodine. So, after the reaction is over, and the starch broken apart, you will only see the original yellowish brown color of the iodine itself.


 Who's ready to explore their Spit? 



1. In a test tube collect about one inch of saliva. This can be combined from lab partners.  This is your tube containing 100% saliva.


2. The 10% Dilution. Fill a graduated cylinder to the 18 ml mark with distilled water. Use a clean transfer pipette to pipet 2 ml of 100% saliva from the bottom of the tube to the water in the cylinder; the total volume in the cylinder will then be 20 ml. Pour the contents of the cylinder and a 50 ml beaker 5 times to mix thoroughly - end with the mixture in the beaker. Label the beaker10%.


3. The 5% Dilution. Pour 10 ml of water into the cylinder.  Add 10 ml of the 10 % dilution (which you just made) to the water in the cylinder; again the total volume should be 20 ml. Pour the mixture back and fourth between the cylinder and an unused beaker until it is thoroughly mixed. Label the beaker 5%.


4. The 2% Dilution. Pour 12 ml of water into the cylinder.  Add 8 ml of the 5% dilution. Mix as before, using a clean beaker. Label the beaker 2%.


5. The 1% Dilution. Pour 10 ml of water into the cylinder. Add 10 ml of the 2% dilution. Mix as before, using another clean beaker. Label the beaker 1%.


This table summarizes the dilutions you are making:




In this part, you will determine whether the speed of the reaction depends on the concentration of the enzyme.


1. Label 4 clean test tubes 10%, 5%, 2% and 1%, Store them in a row in the test tube rack.

2. Pipet 2 ml of each dilution to the corresponding tube.


3. Obtain 4 more test tubes. Pipet 2 ml of starch to each tube. 


4. Place these tubes in a row directly behind the tubes of enzyme.

5. Place one drop of iodine reagent into each well of the spot plates.


6.  Pour the tube of starch into the 2 ml of the 10 % saliva and exactly 10 seconds later take out one drop with the appropriate pipette and drop it in a well of iodine reagent. 


Repeat the same test every ten seconds until the starch is completely digested in the test tube.


Make sure to take a fresh drop from the tube for every 10 second test. 


Figure out how many seconds it took for the salivary amylase to degrade the starch completely. 


Record results in a data table. 


Reaction rate versus Enzyme Concentration

Reaction Time in Seconds






7. Repeat with each of the three other saliva dilutions.




Now you will study the same reaction at four different temperatures; very cold (iced conditions), room temperature, body temperature (37) and very hot (hot water bath 80).


1. Examine your data from part 2. Decide which concentration of saliva had a reaction rate longer than 10 seconds (probably 10% or 5%). From now on, use this dilution for the rest of the experiments. Use a pipette to transfer 1 ml of this concentration into each of 4 clean test tubes.


2. Make four tubes of starch solution as above.


3. Set one tube of diluted saliva and one tube of starch into a beaker of ice and water. Leave them there at least 5 minutes. Meanwhile clean and prep a spot plate with iodine.


4. Mix the starch into the saliva. Test immediately for the presence of starch every 30 seconds (not 10), until the test is no longer positive. 


Record. If the enzyme has not completely digested the starch in 3 minutes, the enzyme is nonfunctional. In this case record inactivated enzyme.   Reaction rate versus Temperature


5. Repeat steps 3 and 4 using the other temperatures. Make sure and let the unmixed tubes sit in the appropriate temperatures for at least five minutes before mixing them.


Post lab question:

Subject Guide

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Jennifer Jourdain
Montachusett Regional Voc Tech School
1050 Westminster Street
Fitchburg, MA 01420
(978) 345-9200 x5125