Caught by a Kiss
The case: A number of pieces of potential evidence were collected from
the scene of a crime: a drinking cup, a toothpick, a drinking straw, and an
envelope that had been sealed. The Crime Scene Technician (CST) assigned to
the case has asked that the laboratory analyze the items for the presence of saliva
by looking for amylase activity, in anticipation of subjecting the evidence to DNA testing.
Background: Enzymes are a unique class of proteins because they can speed up or catalyze other
reactions without their own structure being changed. Enzymes participate in many functions in our bodies,
from helping to digest food we eat to making copies of our DNA each time our cells divide. In this case, the
focus is on the activity of the enzyme amylase. This is the main digestive enzyme found in human saliva.
Amylase catalyzes the breakdown of starch into shorter disaccharides. Amylase is a fairly universal enzyme,
being well conserved across species that use starch for energy (including beans, wheat, flies, and mammals).
Humans have two amylase genes, one that is active in the pancreas and another in the salivary glands. The
presence of amylase in saliva appears to be a fairly recent evolutionary development, since many other
organisms only produce amylase in the pancreas. Forensic scientists use the fact that saliva contains amylase to
determine whether saliva is present on a piece of evidence. Saliva found at the crime scene is helpful to the
forensic scientist because cells from the epithelial lining of the mouth are frequently sloughed off into the
saliva. Thus saliva can be a source of DNA, which can further identify an individual.
Amylase activity is very easy to detect. Most assays to detect amylase rely on a simple reaction, where iodine
reacts with starch to produce a purple color. Because amylase degrades starch, and iodine will not react with
degraded starch, a purple color reaction is not seen when amylase activity is present. These reactions can take
place on an agar plate containing starch, or any area where starch is present.
In this case, a number of pieces of evidence have been isolated from the crime scene. You are to determine if
any of the pieces of evidence contain saliva. If so the piece of evidence is valuable because it contains cells that
hold DNA that could be used to identify individuals involved in a crime.
Pre-lab questions:
Please place your answers to these questions in the spaces below…..complete sentences are not required.
1. What has the CST asked the lab to do for them?
2. What is an enzyme? Which enzyme is the focus of this investigation?
3. What is the function of the enzyme from #2? Where in humans is it found?
From Carolina Biological Supply Company Caught by a Kiss 21-2015
4. How do forensic scientists use the fact that saliva contains amylase? How is it helpful?
5. What do you think an assay is?
6. If amylase is present (saliva) what will be the color noted at the end of step 8 on the agar plate? Why?
7. If amylase is not present (no saliva) what will be the color noted at the end of procedure step 8 on the
agar plate? Why?
Procedure:
1. Locate the “starch agar positive/negative” control plate. Observe what will be seen for a positive and
negative test.
2. Obtain a starch agar plate that you will use to test your evidence. Using a marker, on the bottom of the
plate, mark off four sections. Label each section with the piece of evidence you will be testing in that
particular section.
3. Get the piece of evidence you want to test. DO NOT allow one piece of evidence to come in contact
with another piece. Preventing contamination is a large part of the forensic scientist’s job.
4. Prepare a cotton ball/Q- Tip by wetting lightly with water. DO NOT soak the cotton ball, and press out
any excess water against the side of a container/sink.
5. Rub the wet cotton ball/Cue Tip several times over an area of the piece of the evidence where you
suspect the presence of saliva. When doing this, make some distinctive mark so will know for certain if
a reaction occurs.
6. Using a different applicator, isolate some human saliva and rub it in the + area of the plate.
7. Allow the plate to incubate at room temperature for 15 minutes. This allows time for the amylase to
digest the starch in the Petri plate. Make sure you have your name on your plates as well.
8. “Flood” the plate with IKI solution. Observe any color change.
From Carolina Biological Supply Company Caught by a Kiss 21-2015
Data Analysis
This is to be completed at the end of the activity. It MUST be typed. Of course all answers
must be done in complete sentences.
1. Prepare a written report for submission to the Crime Scene Technician regarding what pieces of
evidence most likely contain saliva and thus DNA. Include a description of the technique used to
determine this data and any information you may have deduced regarding the concentration of saliva on
the particular object.
2. What results appeared on the “starch agar positive/negative” control plate?
3. Why was it important that you look at this plate before beginning your investigation?
From Carolina Biological Supply Company Caught by a Kiss 21-2015
Caught by a Kiss – Teacher Prep
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Prep the plates the day before. You can either have the students make a quad on the lid and use one plate
for 4 pieces of evidence OR have one plate per evidence.
Prep a control plate. Spit on to the agar, incubate for 15 minutes. Saturate with IKI on the spit and on
non-spit areas.
o Starch + iodine  blue color
o Starch (which is a polysaccharide) + amylase  disaccharides
o Disaccharides + iodine  clear to yellow color
Tell the kids that with crime evidence, first we need to know if there is a possibility of DNA. So we will
test for saliva (which cheek cells could be in and those cells could have DNA)
Results may be obtained after approximately 10 minutes of incubation in many cases.
In preparation of the evidence, each piece should be in its own bag. Each piece of evidence does not
need to be prepared with saliva but YOU will need to know which samples are or are not.
The incubator should be at 37 degrees C.
When steaming the envelope, you will need a hot plate and a beaker of water. Do not allow the saliva
residue to become too hot. Amylase is an enzyme fairly resistant to heat, but it may lose activity or
denature if left too long in the steam.
Iodine – dilute the IKI solution 1:10 in distilled water by mixing 30 mL of IKI solution with 270 mL of
distilled water. This is the working stock.
Discuss the amylase enzyme and the starch/iodine test as described in the Background to familiarize
students with the enzyme and the activity.
Should take 55 minutes.
Each team will need:
 4 pieces of crime scene evidence
o Straw, cup, toothpick, envelope (or anything else that could hold/have saliva like pen tops)
 3-4 starch agar plates
 6-8 cotton balls or Q-tips
 30 mL of Potassium Iodide (IKI) solution
 Lab marker (to mark on the dish)
 Water
Safety
 Iodine stains
 Hot plates with hot water
 Dispose of used plates under haz-mat protocol.
From Carolina Biological Supply Company Caught by a Kiss 21-2015