TEACHER INSTRUCTIONS

Goals
1. Introduce students to some of the techniques used by forensic scientists for analyzing blood.
2. Introduce students to the concept of blood type.
3. Provide opportunity for students to practice critical thinking skills in the context of scientific inquiry.

Materials
- Blood (chicken or cow; can be obtained from meat packing facility)
- Red paint
- Red food colouring
- Fresh tomato
- Fresh, raw beet
- Canned tomato sauce or spaghetti sauce
- Small plastic containers in which to aliquot each of the above substances (6 per student group)
- Set of envelopes containing cotton squares with stains from crime scene (one set per student team)
- Small dropper bottles or small plastic tubes and pipettes or droppers
- Hydrogen peroxide
- Phenolphthalein solution (instructions for prep immediately below)

Stock solution
Combine:
- 2 g phenolphthalein (powder)
- 20 g potassium hydroxide (CAUTION: caustic, strong base)
- 100 ml water.
Mix thoroughly.

Add:
- 20 g powdered zinc.
Allow 48 hours for solution to become colourless. Store in brown bottle or bottle wrapped with foil.

Working solution:
- 20 mL stock solution
- 80 mL ethanol

"Case of the Hacked High Tech Lab"
- 1 envelope with dry cotton square stained with red dye labeled "A"
- 1 envelope with dry cotton square stained with cow or chicken blood labeled "B"

NOTE: Rather than providing the students with stains to test, you may have them test the actual stains they collected from the crime scene.
- Simulated Blood Typing Kit with samples transferred to new tubes labeled "suspect 1", " suspect 2", " suspect 3", and "evidence". Place liquid from the same sample in both tubes " suspect 1" and "evidence" (e.g. Mr. Smith from the Wards kit). Place liquid from individuals with different blood types in tubes "suspect 2" and "suspect 3".

Instructions
This activity contains two parts.

Part One is intended to teach students about the catalase test for the presence of blood. While there are more sensitive tests for the presence of blood that an investigator might use, this is by far the cheapest. Following the student handout should be fairly straightforward. Students predict whether or not the substances provided will be catalase positive or negative, then they test their predictions. They also test whether each substance tests positive for blood using the phenolphthalein test. After this step they open the evidence packets provided, and test whether each stain that was found is likely to be blood.

Part Two addresses blood typing. A good way to avoid using actual human blood for this exercise is to purchase a simulated blood typing kit from a biological supply company. Their price range is approximately $35-$50.

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STUDENT INSTRUCTIONS

Investigators often find blood stains during their examination of a crime scene. They also find stains that could be either blood or some other similar substance, like reddish-brown paint. What other things can you think of that might look like blood? How would you test a stain to see if it is blood?

Have you ever used hydrogen peroxide to clean a cut or a scrape? What happened when the hydrogen peroxide came in contact with the blood from the wound?

Blood contains an enzyme called catalase, which breaks down hydrogen peroxide into water and oxygen gas.
2H2O2 --- catalase --> 2H2O + O2

When this reaction occurs, the oxygen gas is released as bubbles. The catalase enzyme performs an important function to living organisms because hydrogen peroxide is very toxic to living cells. Other organisms, including plants and some bacteria, also make catalase.

If you place a few drops of hydrogen peroxide on a substance that contains catalase, it will bubble profusely. These substances that bubble with the addition of hydrogen peroxide are said to test positive for catalase.

Criminal investigators do not typically use the catalase test at crime scenes. Other simple tests are better at detecting very dilute concentrations of blood – sometimes so dilute the human eye can longer see the stain. These tests (listed below), while more reliable, require more expensive chemicals.

- Benzidine
- Leucomalachite green
- Phenolphthalein
- Takayama test
- Tetra-methyl bezidine
- Luminol and Spectrophotometric tests.

Most of these tests rely on the activity of peroxidase enzymes in blood to react with a chemical stain causing it to change colour, or in the case of luminol, glow in the dark.

In this activity, you will be comparing the results of the catalase test using hydrogen peroxide with the phenolphthalein test, to see how each reacts with blood and other substances.

Which of the following substances do you think would test positive for catalase? Make a prediction for each, and explain your reasoning. Make sure you make a prediction for each substance before conducting your test.

Test each of these substances to see if it is catalase positive or negative by placing a few drops of hydrogen peroxide on a small amount of each. Record results in the table above.

SAFETY NOTE: Even though you will not be using any real human blood in this activity, you should wear appropriate protection such as gloves.

Analysis of evidence from the crime scene
Test any stains from the crime scene that you suspect may be blood stains. You should only test part of each sample and not the whole sample. Why?
Record your results below.

Which of these stains is probably blood? Could it be anything else other than blood? Check your answers against the key provided.

Once you know that a stain is blood, what else would you do as a forensic scientist? There is a lot of potential information in a blood stain.

Pattern and shape: The shape and pattern of blood drops can reveal important information about the nature of the wound from which the blood came. Was the bleeding person standing still or walking? What distance did the blood drop fall? Did the blood spatter in all directions? A good investigator would carefully photograph all blood stains from different angles both so that a forensic scientist could examine the pattern and to be able to present the evidence to a jury.

DNA: Blood contains DNA, and depending on the size of the stain and its condition (old, new, dry, etc.), a forensic scientist may be able to get enough information to obtain a highly probable match of a suspect with the evidence.
Two techniques are heavily used by forensic scientists in evaluating DNA evidence from blood or other body tissues – polymerase chain reaction (PCR) and variable number tandem repeats (VNTR’s).

Type: Blood typing can be used as an initial test to exclude some suspected sources of a bloodstain. For example, if a blood stain at the crime scene contains Type A blood, but the key suspect has Type O blood, the suspect could
be excluded as a source of the blood stain – meaning he or she definitely did not leave the blood stain. However, blood type alone usually cannot positively identify a suspect because many people share the same blood type.

Investigators have collected blood samples from each of the suspects in the case. The samples and the evidence are labeled A-D. It will be your job to type each sample. You will determine both the ABO blood type of each sample as well as the Rh factor type.

ABO blood group:
There are three alleles at the locus that determines an individual's ABO blood type, and there are four possible types --> A, B, AB, and O. Type A individuals have "A" antigens in their blood. Antigens are proteins that the body's immune
system recognizes and either mounts an immune response to, if the antigen is from a foreign source, or ignores, if the antigen is part of the body itself. Type A individuals do not mount an immune response against A antigens. If they did, the immune system would produce A antibodies that would bind to the A antigens and cause the blood to thicken and clot. Individuals who are type B don't produce antibodies against B antigens, but they do produce antibodies
against A antigens. Individuals who are type O have neither A antigens or B antigens, so they have antibodies to both types. Individuals who are type AB, have both antigens and do not have antibodies to either A or B. There are no O antigens. Type O individuals simply do not produce any antigens in this blood type group.

The Rh factor:
Another commonly tested blood antigen group is the Rh factor. Individuals who produce Rh antigens are referred to as Rh positive. Individuals who do not produce Rh antigens are referred to as Rh negative.

Follow the directions provided with your blood typing kit and determine the blood types of the samples labeled A, B, C, and D. Remember to wear gloves while handling the blood samples. Record the blood types of each individual below. Consult the key to the labels and write in the identity of each sample.

Answer the following questions regarding your results:
1.) Based on the results of the blood type analysis, can you exclude any of the suspects as having left the blood stain found at the crime scene?
2.) Based on the results of the blood type analysis, which suspect(s) could have left the blood stain at the crime scene?
3.) If you were allowed to perform additional tests using this blood stain from the crime scene, what would you recommend?