Activity 8-1
A Presumptive Test for Blood Teacher Notes
Materials:
Cloth or shirt with dime-sized stain of animal blood
Blood from animal source
Ketchup – about the size of a dime
Two prepared samples with dime-sized ‘blood’ samples
20 ml 3% Hydrogen peroxide solution in dropper bottle
20 ml 95% ethyl alcohol in dropper bottle
20 ml Distilled water in dropper bottle
20 ml 2% phenolphthalein solution in dropper bottle
Biohazard container
Latex or nitrile gloves
Safety precautions:
1. Wear protective gloves.
2. Dispose of all samples in a biohazard container provided.
Data Table 1
Stains
Color
Pink or not pink?
Describe your observations
Is it blood or not blood?
Blood stain
(positive control)
Ketchup
(negative control)
Unknown # 1
Unknown # 2
Questions
1. Complete the following Data Table indicating the role or function of each of the
chemical reagents used in this experiment.
Chemical
1, Deionized Water
Function
Used to help dissolve blood from the stained fabric
2. Alcohol
“cleans” the area so that hemoglobin is more
exposed
3. Phenolphthalein
Serves as the oxygen acceptor
Oxidized phenolthalein turns pink
Color indicator
4. Hydrogen Peroxide
Substrate for the perioxidase-like chemicals found in
hemoglobin
2. Explain the role of using both a positive and negative control group before testing
the unknown stains.
a. Positive control Used to verify that all chemical reagents are functioning in
the expected manner. If the expected color change does not occur using the
positive control, then new reagents are needed before testing any
unknowns.
b. Negative Control Used to verify that a pink color will not result when using
these reagents. If a pink color is produced with the negative control, you
need to get new reagents. There could possibly be some contamination of
your reagents.
3. If animal blood is different from Human blood, how it is possible to get a positive
reaction with the Kastle-Meyer test using dog blood?
The Kastle-Meyer Test detects the presence of Heme found in hemoglobin of the
blood. Both a dogs and humans have hemoglobin in their blood. Therefore, both
dog and human blood will produce a positive reaction using the Kastle-Meyer test.
4. List two types of materials that might produce a false positive test when performing
the Kastle-Meyer test for the presence of blood.
A. Raw vegetables such as potatoes, beets and horseradish contain
perioxidases.
B. Animal blood will produce a positive reaction using the Kastle-Meyer
tests.
5. Why is it important to use a cotton swab when doing this test? Why aren’t the
reagents applied directly to the original blood stain?
It is important to preserve the evidence and avoid any contamination of the
original piece of evidence. The bloody fabric may need to be reexamined at
a later date. Therefore, avoid adding anything to the original stained fabric.
6. Suppose that a red stain was found in a bathtub along with some bath water.
Would it be possible to detect the blood since it might have been diluted?
The Kastle-Meyer blood test is sensitive to dilutions of one part blood to 10,000s
parts water. If the dilution of the blood does not exceed this level, then the blood
could indicate a positive reaction. The more diluted the blood, the lighter the
reaction color.
7. When should the pink color first be evident:
a. When applying the phenolthalein to the cotton swab or
b. When applying the Hydrogen Peroxide to the cotton swab
Explain your answer.
The pink color should only be visible after adding the Hydrogen Peroxide. If the
pink color appears after adding the phenolthalein, something is wrong. A new test
should be performed. Recall the pink color occurs after the oxygen is released from
the Hydrogen Peroxide. It is the heme molecule that acts as the perioxidase
causing the Hydrogen Peroxide to break down. If the Hydrogen Peroxide is not yet
added, there is extra oxygen already present.
Further Research
1. Research the history of using this method as a presumptive blood test. Investigate
the role of each of the scientists:
a. Louis-Jacques Thenard discovered Hydrogen Peroxide in 1818
b. Christian Freidrich Schonbein developed one of the first presumptive tests in
1863
c. Dr. Kastle Early 1900’s developed a presumptive test for hemoglobin using
phenolthalein
d. Dr. Meyer later refined and improved on Dr. Kastle’s test
2. Research how to distinguish dog blood from human blood.
To determine if blood is human or blood from some other animal, a
Precipitin test is performed. Essentially, this test utilizes an antigen, antibody
response. This test was developed in Germany in 1901 by Paul Uhlenhuth.
To determine if the blood is human, it is necessary to produce anti-human
antibodies. Injecting some human blood into a different animal, a rabbit for
example does this. When human blood is injected into the rabbit, the rabbit
produces anti-human antibodies or anti-serum.
To perform the precipitin test, a sample of the suspected human blood in
placed in the depression of a gelatin coated depression slide. Anti-human
antibodies or anti-serum if placed on the opposite side of the depression
slide. After applying an electric current, the protein molecules in the two
samples migrate towards each other forming a precipitin line where the
proteins combine (antigen and antibodies).
To determine if the blood sample was from a dog, a precipitin test would be
performed. To determine if the blood came from a dog, some of the dog’s
blood would be injected into a rabbit so that the rabbit produced antibodies
or anti-serum against the dog blood. Once these anti-dog antibodies are
formed they would be combined with a sample of the suspected dog blood.
If a precipitin line forms, this indicates that the blood was from a dog.
Dog’s blood contains over 13 different red blood cell antigens.
Over 13 canine blood groups have been described. Eight DEA (Dog
Erythrocyte Antigen) types are recognized as international standards.
<ref>Symons M, Bell K. ''Expansion of the canine A blood group system.''
Anim Genet 1991;22(3):227-35.</ref>
<ref>Symons M, Bell K. ''Canine blood groups: description of 20
specificities.'' Anim Genet 1992;23(6):509-15.</ref>
<ref>Andrews GA, Chavey PS, Smith JE
3. Investigate the presumptive test for the presence of semen. Describe how this test
is performed.
Semen evidence may not be visible to the naked eye. However, with a black
light semen may appear as white or blue light. Because urine may also
produce the same color it is not possible to be certain if the stain is from
Urine or semen.
If the stain is examine under a microscope, it would be possible to view
Sperm. A limitation of this test is that not all men produce sperm, or, may
Produce sperm in such low numbers that the sperm may not be detected.
Semen can be chemically analyzed for the presence of Acid Phosphatase.
This is an enzyme in high concentrations in semen. The stain is treated with
a mixture of Calcium alpha-naphthyl phosphate and II-naphthylin diazo bluB. If the stain turns a purplish-glue color, then the stain contains semen.
1. If a man had a vasectomy, is it possible to check the examine bed sheets for the
presence of semen. Explain.
Yes, Sperm are not necessary to confirm the presence of semen. As
described above, a chemical test testing for the presence of Acid
Phosphatase found in semen can be performed.
2. Famous Case Histories involving Blood. Research each of the following cases.
Explain the role of blood in helping to solve the crime.
a. Ludwig Tessnow Case
Known as the “Monster of Rugen or the Mad Carpenter of Rugen”
(Germany)
July 1, 1900, Ludwig murdered and mutilated two young boys. He tried to
Tell the Judge that the red stain was from a wood dye used during his
Work with carpentry. However, Uhlenhuth had just published a paper dated
Aug. 1, 1900 describing how he could tell the existence of blood stains and
the difference between human blood and animal blood. It was found that
the red stain was not from carpenter’s dye, but was indeed blood. Tessnow
was sentenced to death.
b. O.J.Simpson Case
Blood evidence was found not only at the crime scene, but also on O.J.
Simpson’s white bronco vehicle. This trail of blood linked O.J. Simpson to
the crime scene.
The many crime scene investigators and police who arrived at the murder
scene contaminated Blood evidence.
Blood evidence photographed on the back of Nicole Simpson was later lost
when crime scene investigators turned over her body.
O.J. Simpson was not convicted of this crime. Many people feel that
although the DNA evidence linked him to the crime, the fact that so much of
the evidence was not properly handled and processed led some people to
feel that all of the evidence could not be used against O.J. Simpson.
He was later held responsible for the murders of his wife, Nicole Simpson
and a young man named Ron Goldman in a civil lawsuit.
3. Explain the role of “secretors vs. non-secretors in body fluids.
a. What are secretors?
Eighty percent of the population is known as secretors. Within their body
fluids such as saliva, perspiration, urine and semen are some of the same
substances that are found in their blood.
b. When were secretors first identified? 1925
c. How can the classification of a secretor vs. a non-secretor serve to help
identify a suspect or victim?
This meant that the blood type of an individual could be determined in the
absence of blood as long as some other body fluid was available.