Blood, I Presume?

Stephen Gallagher

Harry S. Truman High School, Bronx

Summer Research Program for Science Teachers

August 2009




How do forensic scientists test evidence for the presence of blood?  Properly collected and preserved blood evidence can establish a strong link between an individual and a criminal act. ABO blood type, for instance, can eliminate certain suspects, while DNA obtained from blood can have enormous probative value. This lesson is part of a unit on Forensic Serology, which involves the study of blood, semen and saliva or sweat in matters pertaining to the law.


Time: 40 Minutes


Bovine blood; blood cards (filter paper pre-stained with bovine blood);  test tubes; eye droppers; scissors; tweezers; cotton swabs; phenolphthalein; hydrogen peroxide; red food dye; lipstick; red marker; clam juice or cranberry juice; miscellaneous items of clothing or objects to be stained as evidence; white paper; bleach; paper towels.

Do Now

Show students a series of pictures of objects with red stains.  Ask, which stains are blood?  How do they know?   Why is important to determine which stains are blood?


Blood is the most common physical evidence in accidents, murder cases, and violent crime investigations. Besides blood, crime scene technicians may also find other stains and residues similar to blood in appearance at the scene, such as tomato sauce, red paint, or animal blood. A number of simple presumptive tests exist to distinguish blood from other stains.

One of the most common tests for the presence of blood is known as the Kastle-Meyer test.  It is based on the idea that hemoglobin increases the ability of hydrogen peroxide to act as an oxidant.  In the presence of hydrogen peroxide, a molecule of hydrogen peroxide (H2O2) oxidizes another molecule of hydrogen peroxide, forming bubbles of oxygen, as follows:


In the early 1900’s, Dr. Kastle developed a presumptive test for the presence of hemoglobin using phenolphthalein’s color change in the presence of oxygen.  In this test, phenolphthalein is reduced and pre-dissolved in alkaline solution, giving it a faint yellow color.  Then, in the presence of hydrogen peroxide in alkaline solution, the hemoglobin catalyzes the oxidation of this form of phenolphthalein to its normal form, which results in a color change from yellow to intense pink.

 Lesson Overview

In this hands-on lesson, students will learn how to distinguish blood from other stains using the Kastle-Meyer test.  They will also determine the sensitivity of the test using serial dilutions of blood. (N.B.:  All or part of these hands-on activities can be incorporated in this lesson.)

In the first part of the lesson, supply each student (or small teams of students) with:

·         a vial of bovine blood

·         a blood card – filter paper pre-stained with bovine blood

·         test tubes, and a test tube rack

·         eye droppers

·         a scissor

·         tweezers

·         cotton swabs

·         phenolphthalein solution

·         hydrogen peroxide

·         water

Students test the reagents using a negative control and a positive control:  Each student should cut a very small piece from the blood card and place it in a test tube.  A few drops of phenolphthalein should be added to this tube (enough to just cover the sample), and to a second test tube that does not contain a blood card clipping.  A few drops of hydrogen peroxide should be added to both tubes, which should then be compared.  Students should note the color changed expected when blood is present.

Students use a cotton swab to test for the presence of blood:  Students apply a few drops of water to the end of  two cotton swab.  They then gently rub one swab against the bovine blood card until the swab picks up some of the blood.  Apply a few drops of phenolphthalein and hydrogen peroxide directly to both swabs.  Note the color change for a positive test result. (Ask students why they tested the other swab. Elicit: It functions as a control group.)

Students use the Kastle-Meyer test on evidence:  Evidence stations are set up around the classroom.  Each station has one item of evidence featuring a small red-colored stain. (These can be created using red food dye and/or marker, lipstick, cranberry juice, or any other substance that creates a red stain.)  Students use their swabs to perform the Kastle-Meyer test directly, and/or take a small clipping from each stain (with scissors and tweezers) and perform the test in a test tube.  (Irrespective of which technique they use, students must include a control group. Also, if the same scissor and tweezers is used at more than one station, or repeatedly at the same station, they must be cleaned with bleach and thoroughly dried between each use.)

Students determine the sensitivity of the Kastle-Meyer test:  Have students make a serial dilution of bovine blood by setting up a test tube rack with six test tubes.  Each tube should be labeled as follows:  1) Neat, 2) 1/10, 3) 1/100, 4) 1/1000, 5) 1/10,000, 6) 1/100,000.   Undiluted bovine blood is placed in the first tube.  (This is known as a “neat” sample.)   Eighteen drops of water are placed in each of the other tubes.  Two drops of blood from the neat sample are then added to tube #2, and gently mixed.  Using a clean eye dropper, two drops from tube #2 are removed and added to tube #3.  This progression is repeated until the serial dilution is completed.  Students now perform the Kastle-Meyer test on each tube by adding phenolphthalein and hydrogen peroxide.  Each tube is then viewed against white paper.  (Which tube did not show a color change?  What is the sensitivity of the Kastle-Meyer test?)


S4d, S4e, S5a, S5c, S5d, S5f, S6a, S6d, S7b, S8a