Experiment 6

Blood Stain Analysis

Jessica Nicholson and Madi Kreitz

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:

Part 1: Test for Catalase using Hydrogen Peroxide

1. A few drops of 3% hydrogen peroxide are placed on a small amount of each substances

smeared/stained on a piece of cloth

2. Record results in table 1

Part 2: Test for Presence of Blood using the KM test kit

1. Add one drop of Kastel-Meyer reagent to select spots

2. Wait a few seconds, note the time

3. Add one drop of 3% hydrogen peroxide to the same exact spot

4. Record results in table 2

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

2. Record results in table 3

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

2. Observe the chemiluminescence in the dark

3. Record results in table 4

Part 5: Test the Sensitivity of combined KM method, sequential KM method and TMB

method

1. Prepare multiple dilutions of blood from 1:100 to 1:10,000,000 in a 12 well plate

2. Place 450 μL water to multiple wells first

3. Then, add 50 μL of synthetic blood to the 1:10 diluted solution

4. Transfer 50 μL to another well containing 450 μL water to get 1:100 dilution, and so on

5. Each dilution is taken and transferred to a paper and allowed to dry

 6. With a pencil, label 4 sheets of copy paper

7. Make 2 to 3 cm circle next to each label

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

Part 6: Examine the Existence of Latent Bloodstains on Washed Clothing

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

should be no color change

4. Apply one drop of hydrogen peroxide to the tip of the cotton swab, observe for a color

change

Chemical Hazards:

 Wear goggles, gloves, and lab coat always

 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 1: Test for Catalase using Hydrogen Peroxide

Substance Do you predict it will Observation Result

catalase + or -?
Red Ink -, There is nothing in red ink No Change -
that would react to hydrogen
peroxide
Fresh Tomato -, There is nothing in fresh No Change -
tomato that would react to
hydrogen peroxide
Cooked -, There is nothing in cooked No Change -
Tomato tomato that would react to
hydrogen peroxide
Red Food -, There is nothing in red No Change -
Coloring food coloring that would
react to hydrogen peroxide
Fresh Raw -, There is nothing in fresh No Change -
Beet raw beet that would react to
hydrogen peroxide
Synthetic +, synthetic blood contains Bubbling and fizzing +
Blood things like iron that will occurred
cause a reaction with
hydrogen peroxide
(Table 1)

Part 2: Test for Presence of Blood using the KM test kit

Substance Do you predict it will Observation Result

catalase + or -?
Red Ink -, There is nothing in red ink -3 Seconds -
that would react to the KM -No pink color
reagent
Fresh -, There is nothing in fresh -3 Seconds -
Tomato tomato that would react to the -No pink color
KM Reagent
Cooked -, There is nothing in cooked -3 Seconds -/+
Tomato tomato that would react to the -Slightly Pink for a
KM Reagent second
Red Food -, There is nothing in red food -3 Seconds -
Coloring coloring that would react to the -No pink color
KM Reagent
 Fresh Raw -, There is nothing in fresh raw -3 seconds -
Beet beet that would react to the -Purple disappears
KM Reagent -No pink color
Synthetic +, synthetic blood contains -3 Seconds -
Blood things like iron that will cause -No color change
a reaction with the KM Regent
(Table 2)
Part 3: Test for the Presence of Blood using the TMB test kit

Substance Do you predict it will Observation Result

catalase + or -?
Red Ink -, There is nothing in red ink -Remained same -
that would react to the TMB color
Reagent
Fresh -, There is nothing in fresh -No color change -
Tomato tomato that would react to the
TMB Reagent
Cooked -, There is nothing in cooked -No color change -
Tomato tomato that would react to the
TMB Reagent
Red Food -, There is nothing in red food -No color change -
Coloring coloring that would react to the
TMB Reagent
Fresh Raw -, There is nothing in fresh raw -No color change -
Beet beet that would react to the
TMB Reagent
Synthetic +, synthetic blood contains -Slightly Green/Blue +
Blood things like iron that will cause color
a reaction with the TMB
Reagent
(Table 3)

Part 4: Test for the Presence of Blood using the Luminol test

Substance Do you predict it will Observation Result

catalase + or -?
Red Ink -, There is nothing in red ink -No Change -
that would react to the luminol
Fresh -, There is nothing in fresh -No Change -
Tomato tomato that would react to the
luminol
Cooked -, There is nothing in cooked -No change -
Tomato tomato that would react to the
luminol
 Red Food -, There is nothing in red food -Bright Blue tint +
Coloring coloring that would react to the
Luminol
Fresh Raw -, There is nothing in fresh raw -No change -
Beet beet that would react to the
Luminol
Synthetic +, synthetic blood contains -Bright blue tint +
Blood things like iron that will cause
a reaction with the Luminol
(Table 4)
Part 5: Test the Sensitivity of combined KM method, sequential KM method and TMB

method

Control Test Observation Time (s) for spot to appear

KM Test + 5 s – 10 s

Sequential KM Test +/- 3 s – 10 s

TMB Test +/- 3 s – 10 s

(Table 5)

KM Test

Blood Concentration Observation (+ or -) Time (s) for spot to appear

1:10 +, bright pink 5 seconds

1:100 +, bright pink 5 seconds

1:1000 False + 3 Minutes

1:10000 False + 3 Minutes

1:100000 False + 3 Minutes

1:1000000 False + 3 Minutes

1:10000000 False + 3 Minutes

(Table 6)
Sequential KM Test

Blood Concentration Observation (+ or -) Time (s) for spot to appear

1:10 +, bright pink 3 seconds

1:100 +, bright pink 3 seconds

1:1000 False + 3 Minutes

1:10000 False + 3 Minutes

1:100000 False + 3 Minutes

1:1000000 False + 3 Minutes

1:10000000 False + 3 Minutes

(Table 7)
TMB Test

Blood Concentration Observation (+ or -) Time (s) for spot to appear

1:10 +, green color 5 seconds

1:100 +, green color 5 seconds

1:1000 False + 3 Minutes

1:10000 False + 3 Minutes

1:100000 False + 3 Minutes

1:1000000 False + 3 Minutes

1:10000000 False + 3 Minutes

(Table 8)
Luminol Test

Blood Concentration Observation (+ or -) Time (s) for spot to appear

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

 Bright blue result

 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

intense detection capabilities of the Luminol.

Work Cited:

Huynch, L., Henck, C., Saxton, K., & Wang, J. (n.d.). Introduction to Forensic Chemistry:

Laboratory Manual (Vol. 1). University at Albany.