Kastle Meyer Test for Blood

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Kastle Meyer Test for Blood
The Kastle Meyer test for blood is a presumptive test. It is highly sensitive, but does not identify the species (human or not).

The Kastle Meyer test for blood is a presumptive test that changes color in the presence of hemoglobin from red blood cells. American chemist Joseph Kastle invented a crude version of the test in 1901, while German chemist and physician Erich Meyer modified and improved the test in 1903.

Here is how to perform the Kastle Meyer test and prepare the reagents and interpret the results. Also, learn how the test works so you can minimize false positives and false negatives.

How to Perform the Kastle Meyer Test

The Kastle Meyer test is inexpensive and simple:

  • Kastle-Meyer solution
  • 70 percent ethanol
  • distilled or deionized water
  • 3 percent hydrogen peroxide
  • cotton swabs
  • dropper or pipette
  • dried blood
  1. Moisten a swab with distilled or deionized water. Alternatively, dampen the swab with alcohol.
  2. Touch the suspected blood sample with the swab. Swabbing or rubbing is unnecessary because of the high sensitivity of the test.
  3. Optional: Apply a drop or two of 70% ethanol to the swab. The alcohol breaks open the blood cells and exposes the hemoglobin, improving test sensitivity.
  4. Apply a drop or two of Kastle-Meyer solution to the the swab. Initially, the solution is colorless or pale yellow. It should not discolor the swab. If the drops are pink the solution is old (oxidized) and will not work. If the swab immediately changes color upon Kastle-Meyer solution application, it either means the solution is old or else there is an oxidizing compound present. In either case, re-test using fresh Kastle-Meyer solution.
  5. Wait a few seconds. Now, apply one or two drops of 3% hydrogen peroxide. An immediate color change to pink is a positive test for blood. An eventual color change occurs whether blood is present or not.
The baking soda and vinegar chemical reaction makes sodium acetate, water, and carbon dioxide.

Color Change Volcano

Do you have leftover materials from the Kastle Meyer test? See how phenolphthalein indicator makes color changing lava in a chemical volcano.

How to Make Kastle Meyer Solution

Kastle Meyer solution is available for purchase, but it’s also easily prepared.

  • 0.1 g phenolphthalein powder
  • 25% w/v sodium hydroxide solution (aqueous)
  • 0.1 g mossy zinc
  • distilled water
  • 70% ethanol
  1. Dissolve 0.1 gram of phenolphthalein into 10.0 milliliters of 25% sodium hydroxide solution.
  2. Add 0.1 gram of mossy zinc. This turns the solution bright pink.
  3. Gently boil the solution until it changes color from pink to either colorless of else pale yellow. Add water as necessary so the volume remains unchanged.
  4. Cool the solution, decant the liquid, and dilute it to 100 milliliters final volume with 70% ethanol.
  5. Store Kastle Meyer solution in a tightly-sealed brown or blue bottle.

How the Kastle Meyer Test for Blood Works

The Kastle Meyer test responds to the iron in hemoglobin of red blood cells. Basically, the iron and hydrogen peroxide oxidizes phenophthalin (the colorless form of the indicator in the test solution) into phenolphthalein (the pink form of the indicator).

heme iron Fe4+ + phenolphthalin (colorless) + H2O2 → phenolphthalein (pink) + H2O + heme iron Fe3+

False Positives and False Negatives

The Kastle Meyer test is a presumptive test, which means a negative result rules out the presence of blood, while a positive test indicates a sample is probably blood. However, a positive test result really only indicates the presence of hemoglobin, which comes from red blood cells in mammals and other animals. Bacteria and even plants can produce hemoglobin under the right circumstances. The Ouchterlony test is one of the tests that determines whether blood is human in origin.

Additionally, certain situations lead to false positive and false negative results.

  • Copper salts, nickel salts, and other chemical oxidants turn the indicator pink before the addition of hydrogen peroxide. Test for these substances by waiting a few seconds between the addition of the Kastle Meyer solution and the hydrogen peroxide.
  • Peroxidases from some plants give a false positive test result. Examples of plants containing these compounds include broccoli, cauliflower, and horseradish. These plants are great substitutes for real blood in a classroom demonstration!
  • Reading the test result more than 30 seconds after the addition of hydrogen peroxide generally produces a false positive. This is because the indicator oxidizes in air on its own within this amount of time.

Other Limitations

The test is sensitive enough that it detects blood diluted down to 1:107. This is roughly one drop of blood in 10,000 drops of water. While this pretty dilute, trace quantities of blood might not cause the color change.

The original test uses a swab dampened with water. Treating the swab with ethanol lyses the cells and improves the sensitivity of the test. Theoretically, the test is nondestructive, meaning you can use the blood on the swab for further analysis. However, the sodium hydroxide solution actually does degrade DNA. The usual practice is taking a separate blood sample for analysis, if possible. An article by Sloots et al. in Forensic Science International describes the DNA damage and ways to avert it.

References

  • Gaensslen, Robert E. (1983). Sourcebook in Forensic Serology, Immunology, and Biochemistry. Washington, D.C.: U.S. Government Printing Office.
  • Kastle, Joseph H.; Shedd, Oliver March (1901) “Phenolphthalin as a reagent for the oxidizing ferments“. American Chemical Journal. 26 (6) : 526–539.
  • Meyers, Thomas C. (2006). “Chapter 21: Serology”. In Wecht, Cyril H.; Rago, John T. (eds.). Forensic Science and Law: Investigative Applications in Criminal, Civil, and Family Justice. Boca Raton, FL: CRC Press. pp. 410–412. ISBN 0-8493-1970-6.
  • Ponce, Ana Castelló; Pascual, Fernando A. Verdú (1999). “Critical Revision of Presumptive Tests for Bloodstains“. Forensic Science Communications. 1(2): 1–15.
  • Remsen, Ira; Rouiller, Charles August (eds.) “Phenolphthalin as a reagent for the oxidizing ferments”. American Chemical Journal. 26 (6) : 526–539.
  • Sloots, James; Lalonde, Wendy; Reid, Barbara; Millman, Jonathan (2017). “Kastle-Meyer blood test reagents are deleterious to DNA”. Forensic Science International. 281: 141-146. doi:10.1016/j.forsciint.2017.10.006