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10/26/2017
Mike Miller
Introduction:
Contrary to popular belief, bloodstains are not always obvious and apparent to the
forensic scientist. There are many tests to go about figuring out what is blood and what is just a
mere stain. The first test we are working with is the Kastle-Meyer Test. This test was introduced
in 1901, it uses an alkaline phenolphthalein solution to react with compounds found in blood.
The KM reagent will react with the heme component of the blood (iron contents) and hydrogen
peroxide to produce the oxidized phenolphthalein form, which turns bright pink. This can be
performed two separate ways. We use the secondary version of this method, phenolphthalein
added first with hydrogen peroxide added after. The next test is the Tetramethylbenzidine test
(TMB for short). This is a color test that is safer and more widely used than the previous, the
positive color usually ranges from orange to green. The last test we are looking at is Luminol.
For this test an aqueous or alcoholic solution of luminol and an oxidizer are catalyzed by the iron
present in the hemoglobin component of blood to produce 3-APA in its excited state. We spray
the luminol on the blood and it will create a bright blue outcome if it is positive. All three of
these methods are presumptive tests, the presence of blood cannot be confirmed off of a positive
result.
Experimental Procedure:
1. A few drops of 3% hydrogen peroxide are placed on a small amount of each substances
Part 3: Test for the Presence of Blood using the TMB test kit
1. Place a few drops of the TMB reagent on a piece of cloth stained with a small amount of
each substance
Part 4: Test for the Presence of Blood using the Luminol test
1. Spray the Luminol reagent to the spots on a piece of cloth stained with a small amount of
each substance
Part 5: Test the Sensitivity of combined KM method, sequential KM method and TMB
method
4. Transfer 50 μL to another well containing 450 μL water to get 1:100 dilution, and so on
8. Use a clean pipette to withdraw 15 micro liters of the solution and spot it in the circle
9. Add a drop of a test reagent to the blank area, test whether there is false positive color
showing up and how long the reagents show color in the air
10. For a control, each test reagent should be added to a separate paper with no synthetic
blood
11. Last, we do a luminol test and spray the reagent to the spots
1. Moisten the tip of a cotton swab with a drop or two of deionized water
2. Swab the area of the unknown stains with the tip of the swab and place the swab in a
small beaker
3. Apply one drop of KM or TMB reagent to the tip of the first swab and observe, there
4. Apply one drop of hydrogen peroxide to the tip of the cotton swab, observe for a color
change
Chemical Hazards:
None of the experimental materials should be consumed at any time for any reason
Phenolphthalein can burn or irritate skin, keep out of contact with it.
Results:
Part 4: Test for the Presence of Blood using the Luminol test
method
KM Test + 5 s – 10 s
(Table 5)
KM Test
(Table 6)
Sequential KM Test
(Table 7)
TMB Test
(Table 8)
Luminol Test
1:10 + 1 Second
1:100 + 1 Second
1:1000 - n/a
1:10000 - n/a
1:100000 - n/a
1:1000000 - n/a
1:10000000 - n/a
(Table 9)
Part 6: Examine the Existence of Latent Bloodstains on Washed Clothing
(Table 10)
Rusty color stain shows traces of blood
Could not be anything else except for synthetic blood due to previous results
Conclusion:
For part 5 of the experiment we looked at the sensitivity of every method. They rank in
this order from most sensitive to least sensitive: KM Method, Sequential KM Method, TMB
Method, Luminol. For the KM Method, when both reagents are added together, the first two
ratios (as show in table 6) were the only true positives which was the same as the Sequential KM
Method, when time is taken between the addition of each reagent (table 7) but the regular KM
Method showed a darker marking than showed for the Sequential KM Method. The TMB
method (table 8) took longer to show up so was deemed less sensitive than the both KM
Methods. Luminol did not have a reaction with any of the ratios (table 9) so it falls under the
least sensitive. For part 6 of the lab we received a cloth that had an unknown substance, (table
10, unknown 8) to scan this for blood we first dampened it with water and then we performed the
TMB method by swabbing a cotton ball with the reagent and dabbing it on the unknown area.
When this was done our cotton ball turned bright blue/green immediately showing us that blood
was present on our unknown. This sample when collected has to be collected properly and placed
into a sealed container and not touched until it is being tested, a proper chain of custody must be
kept. KM and TMB tests both spontaneously show false positives from about 1 minute to 8
minutes after the test has been performed on the unknown stain. Other possible approaches to
finding blood more affectively at a crime scene would be a reagent that had the sensitivity of the
first KM method but also had the accuracy and capability of the second KM method and/or the
Huynch, L., Henck, C., Saxton, K., & Wang, J. (n.d.). Introduction to Forensic Chemistry: