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Crime Scene Chemistry: The Kastle-Meyer Test for Blood

Difficulty
Time Required Average (6-10 days)
Prerequisites None
Material Availability You will need to order the "Presumptive Blood Test" kit online. See the Materials and Equipment list for details.
Cost Average ($50 - $100)
Safety Be sure to wear safety goggles and disposable gloves when working with the substances and blood kit.

Abstract

Police detectives use various scientific tools to analyze evidence at a crime scene. One of the classic tools is the Kastle-Meyer test for the presence of blood. This test is inexpensive, easy to perform, and provides quick results. The test provides evidence if red spots found at a crime scene are actually blood. But the investigator needs to be careful, since other substances can also give a positive result. In this crime scene chemistry science project, you will learn how to perform the Kastle-Meyer test for blood, and you will test various substances to see which others also give a positive test result.

Objective

Investigate the chemistry of the Kastle-Meyer test for the presence of blood.

Credits

David B. Whyte, PhD, Science Buddies

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Last edit date: 2013-09-30

Introduction

The Kastle-Meyer test is often used in television crime dramas to show the presence of blood at a crime scene. Evidence that appears to be blood is tested to determine if it is actually blood, and not something that just looks like blood. A spot that might be blood is wiped with a cotton swab to collect some of the substance. A drop of phenolphthalin reagent is added to the sample, and after a few seconds, a drop of hydrogen peroxide is applied to the swab. Sometimes the swab is first treated with a drop of ethanol in order to break open the cells that are present, resulting in increased sensitivity and specificity. This test is nondestructive to the sample, which can then be kept and used in further tests at the lab, such as in DNA analysis. If the swab turns pink rapidly, it is said to test positive for blood. The test result is actually presumptive positive, meaning it is not a conclusive test for blood, and other analyses would typically be carried out to confirm the presence of blood. Waiting for a period of time over 30 seconds will result in most swabs turning pink naturally as they oxidize on their own in the air. Certain chemicals and biological fluids that do not contain blood can also cause the color change. These substances have to be avoided since they produce false positive results; that is, a positive result (color change) in the absence of blood.

The Kastle-Meyer test relies on the iron in hemoglobin, which is the iron-containing portion of a red blood cell, to promote the oxidation of phenolphthalin to phenolphthalein. Phenolphthalin is colorless, but in the presence of blood and hydrogen peroxide, it changes to phenolphthalein, which makes the solution pink. The names of the two chemicals—phenolphthalin and phenolphthalein—are very similar, but they are structurally different. Phenolphthalin is a special form of the common indicator phenolphthalein. It is made by treating phenolphthalein with zinc, which is a reducing agent. In other words, phenolphthalin is made by reducing phenolphthalein. Phenolphthalein, on the other hand, can be made by oxidizing phenolphthalin. The chemistry involved in the Kastle-Meyer test is complex, but the process can be simplified, as shown in Equation 1, below. This equation describes how phenolphthalin (colorless) is oxidized by hydrogen peroxide and heme (which comes from the blood cells) to form phenolphthalein (pink) and water.



Equation 1:
heme iron Fe4+ + phenolphthalin (colorless) + H2O2 → phenolphthalein (pink) + H2O + heme iron Fe3+
  • Heme iron Fe4+ = heme complexed with iron that has four positive charges
  • Phenolphthalin = colorless indicator molecule
  • H2O2 = hydrogen peroxide
  • Phenolphthalein = pink indicator molecule
  • H2O = water
  • Heme iron Fe3+ = heme complexed with iron that has three positive charges

This test is very sensitive. One drop of blood diluted in 10,000 drops of water can still be detected by the Kastle-Meyer test. In this chemistry science project, you will learn about the Kastle-Meyer procedure and test various substances to determine which give a positive Kastle-Meyer test. This will enable you to know what kinds of substances might give false positive results at a crime scene.

Terms and Concepts

  • Kastle-Meyer test
  • Phenolphthalin reagent
  • Positive test result
  • Presumptive positive test result
  • Oxidize
  • False positive result
  • Iron
  • Hemoglobin
  • Catalyst
  • Phenolphthalin
  • Phenolphthalein
  • Indicator
  • Reducing agent
  • Positive control

Questions

  • What is the substance in blood that causes the color change in the Kastle-Meyer test?
  • How sensitive is the Kastle-Meyer test?
  • Based on your research, what substances can cause a false positive Kastle-Meyer test result?
  • Will the test be positive for non-human blood?

Bibliography

This site describes how important it is to make sure that tests like the Kastle-Meyer test are performed correctly.

You could also search the web for videos demonstrating the Kastle-Meyer procedure with actual evidence.

Materials and Equipment

  • Kastle-Meyer Presumptive Blood Test; available from an online supplier such as Carolina Biological, item # 840582. Note: Due to shipping restrictions, this kit must be ordered with the help of a teacher and shipped to school.
  • Substances to test, including:
    • Ketchup (1 bottle)
    • Fresh potato (1)
    • Fresh horseradish (1 piece)
    • Red food coloring (1 small container)
    • Fresh chicken (1 lb. or less—you only need the juice from the meat tray)
    • Fresh beef (1 lb. or less—you only need the juice from the meat tray)
  • Paper plates, small (24)
  • Permanent marker
  • Disposable gloves, can be purchased at a local drug store or pharmacy, or through an online supplier like Carolina Biological. If you are allergic to latex, use vinyl or polyethylene gloves.
  • Knife
  • Cutting board
  • Liquid measuring cup, metric
  • Eye dropper
  • Timer
  • Lab notebook

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Experimental Procedure

Caution: Even though you will not be using any real human blood in this activity, you should wear appropriate protection, such as gloves. Raw chicken could have salmonella. Use gloves and wash your hands after handling chicken and all of the other substances.

  1. Begin by researching the Kastle-Meyer procedure, and carefully read the instructions that accompany the kit.
  2. The kit contains strips of paper that have been dipped in cow blood. These are the positive controls.
  3. Using the permanent marker, label seven plates, as follows: Ketchup, Potato, Horseradish, Dye, Chicken, Beef, and Negative.
  4. Prepare each test substance, as described below, and then add a few drops of each test substance to the middle area of the appropriate plate, using the eye dropper if necessary. Put on your disposable gloves.
    1. For the potato, use a clean knife to cut a piece on a cutting board, then squeeze it directly onto the appropriately labeled plate to obtain fresh juice for testing.
    2. Repeat step 4.a. with a clean knife and cutting board for the fresh horseradish.
    3. For the red food coloring, add 1 drop to 100 mL of water in a liquid measuring cup, then use the clean eye dropper to transfer a few drops to the plate. The red dye presents a challenge because of its color. Through careful dilutions and close observation, you should be able to determine if it causes a positive result or not.
    4. For the chicken and beef, pour a few drops from the container holding the fresh meat onto the appropriately labeled plates. Note: Do not use all the juice, as you will need more for the remaining two trials.
    5. Leave the negative plate empty.
  5. Predict which of the test substances will test positive in the Kastle-Meyer test, and explain your reasoning in a data table, like the one below, in your lab notebook.

Substance Prediction:
Positive or negative?
Explain your prediction. Lab Observations Lab Result:
Positive or negative?
Positive control strips    
Negative control     
Ketchup    
Juice from fresh potato    
Juice from fresh horseradish    
Red food coloring    
Juice from fresh, raw chicken    
Juice from fresh, raw beef    
  1. Following the directions that came with the kit, test each substance to see if it gives a positive Kastle-Meyer result.
    1. Record your observations.
    2. Note that the color should form after the hydrogen peroxide is added, and it should form within about 30 seconds after adding hydrogen peroxide.
    3. For the negative control, rub the swab on the empty plate and treat as above. The negative control demonstrates that there is nothing on the swab or the plate that produces a positive result.
  2. Record your results in your data table.
  3. Repeat the entire procedure, with clean materials, two more times. Record your results in the table.
  4. Which substances gave positive results and which gave negative results? Explain why.
  5. If the results are inconsistent (such as one positive and two negative for one substance in different trials), repeat the test carefully. Keep careful notes that might help explain inconsistent results.
  6. For your presentation, list the chemicals (ethanol, hydrogen peroxide, phenolphthalin) that were used in the test, and the purpose of each.

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Variations

  • Determine how sensitive the test is for the substances that resulted in positive Kastle-Meyer results. Dilute the liquids at various ratios, such as: 1:10, 1:100, 1:1,000, etc. What is the highest dilution at which you still get positive results?

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