PRELIMINARY TEST FOR BLOOD

OBJECTIVE:
To study different preliminary test for blood.
MATERIALS:
Alleged bloodstain, benzidine reagent, hydrogen peroxide solution, Phenolphthalein
reagent, glass slides.
PROCEDURE:
A. PREPARATION OF REAGENTS:
1. BENZIDINE REAGENT:
Warm on a water -bath 13 ml of glacial acetic acid. Dissolve 1.5 gms. of Benzidine crystals in
the warm acid. Add 57 ml of distilled water to this mixture
2. HYDROGEN PEROXIDE: Prepare a 3% solution of this reagent.
3. PHENOLPHTHALEIN REAGENT: Dissolve 2 gms of phenolphthalein and 20 gms of
KOH in 50 ml of HOH to make up a volume of 100 ml. Boil this mixture and then add 10 to 30
gms of powdered zinc. Continue boiling until the solution becomes colorless. Be sure to add
excess zinc to keep phenolphthalein in reduced form (colorless)
B. BENZIDINE TEST: Cut a portion of the blood specimen and put on a clean bond paper.
Add a drop of the Benzidine reagent and a drop of Hydrogen peroxide. Observe any change in
color. Repeat the same test with phenolphthalein reagent. Observe the result. If the stain is a
bloodstain the peroxidase present in the haemoglobin of the blood will carry the oxygen of the
hydrogen peroxide to the benzidine reagent which is then oxidized to produce the blue color.
C. PHENOLPTHALEIN TEST: Soak a portion of alleged bloodstain in a 0.9% saline
solution. After 3 minutes tease the stain and absorb the liquid with a strip of filter paper. Add a
drop of phenolphthalein solution and hydrogen peroxide. In these test the peroxidase carries the
oxygen of hydrogen peroxide to oxidize the colorless phenolphthalein to its colored form.
OBSERVATION:
STAIN BENZIDINE PHENOLPHTHLEIN

STAIN A

STAIN B

STAIN C
CONCLUSION:
1. Which of the alleged stain maybe blood?

2. What is the principle involved in the two test? Explain.

3. With this test, why can’t you conclude that the stain is really blood? Explain.
 CONFIRMATORY TEST FOR BLOOD
OBJECTIVE: To confirm whether or not an alleged bloodstain is really blood or not.
MATERIALS: Known bloodstain alleged bloodstains, which are positive for preliminary test,
saline solution, takayama reagent, glass slides, cover glass and microscope.
PROCEDURE:
A. PREPARATION OF TAKAYAMA REAGENT : Mix 3 ml. of 10% NaOH solution, 3
ml. of pyridine, and 3 ml. of saturated glucose solution and add 7 ml. of distilled HOH.

B. TAKAYAMA TEST: Cut A piece of known bloodstain and place it on a glass slide. Add 3
drops of saline solution and tease with pins. Add two more drop of saline solution and allow
to stand for five minutes. Separate the fibers as a very thin layer and add a drop of the
takayama reagent. Cover with cover glass and view under the microscope and note the
formation of crystals. Repeat the same test with the unknown stain.

OBSERVATION:
1. Describe the color and the shape of the haemochromogen crytals.

2. Draw the haemochromogen crystal you observe.

3. If only stain A produced haemochromogen crystal yet both stain B and C gave positive
result with the benzidine test, you can conclude that both B and C to be bloodstain?
Explain.
CONCLUSION:
1. What is the constituent of the blood, which is responsible for the formation of
haemochromogen crystals?

2. For the purpose of confirming the presence of blood, three methods are used, they are
what?

3. Can takayama test determine whether a bloodstain is human or not? Explain.

4. Give at least four reason which alter the clotting of blood.

5. How many red cells are there in a cubic milliliter of blood?

 PRECIPITIN TEST
OBJECTIVE: To determine whether an unknown bloodstain is human or animal origin.
MATERIALS: Solution of the following dilution:
a. Human blood - 1:2000
b. Cow's blood - 1:500
c. Chickens blood - 1:500
d. Extract of unknown bloodstain -1:2000
e. Extract of unstained clothing 1:2000

Precipitin of required specificity

Normal saline solution
Micro test tube
Micro pipettes
Rack for micro test tube
PROCEDURE: Preparation of Materials
1. 1:2000 dilution of fresh human blood: To 0.5 ml. of fresh blood and saline solution until the
volume is 100 ml.
2. 1:2000 dilution of stain extract: To a few drop of stain extract add saline solution drop by drop
until the bubbles formed after shaking are similar to the size and height of that fresh blood when
also shaken (about 5 minutes).
3. 1:2000 dilution of unstained extract: Place a few drops of unstained extract (same number of
drops as the stained extract) in a test tube. To this add approximately the same volume of saline
used in the preparation of 1:2000 dilution of stained extract.
4. 1:500 dilution of cow's or chicken's blood; Dilute 0.2 ml. of animal blood until the volume is
100 ml.
5. Preparation of Precipitin: (Consult your book)
SOAKING THE STAIN: Cut I square cm. Portions of stained and unstained material into
smaller pieces. Place them into two separate test tube and soak in saline solution overnight.
Transfer the clear liquid into different container and prepare the required dilution for each.
TESTING THE STAIN; To each 8 clean test tube numbered 1 to 8 consecutively, place about
0.05 ml. Of precipitin by means of a micropipette. Centrifuge these tubes and arrange them on
the test tube rack consecutively. Using different pipette; carefully add the following to the
corresponding test tube.
To test tube no. 1 add about 0.05 ml. of 1:2000 dilution of unknown stain no. 1
To test tube no. 2 add about 0.05 ml. of 1:2000 dilution of unknown stain no. 2
To test tube no. 3 add about 0.05 ml. of 1:2000 dilution of unknown no. 2
To test tube no. 4 add about 0.05 of 1:2000 dilution of unknown extract of unknown no. 2
To test tube no 5 add about 0.05 ml. of saline solution
To test tube no. 6 add about 0.05 ml. of 1:2000 dilution of known human blood
To test tube no. 7 add about 0.05 ml. of 1:500 dilution of cow's blood
To test tube no. 8 add about 0.05 ml. of 1:500 dilution of chicken's blood.
Examine the test tube after about five minutes and note the formation of any cloudy
precipitate at the junction of the liquid in each test tube.
OBSERVATION:
In which test tube did you observe white or cloudy precipitate at the junction of two liquid?

CONCLUSON:
1. What are the limitations of precipitin test? Explain.

2. In order that the precipitin must be specified to human what will you do? Explain
 GROUPING OF FRESH DRY BLOOD
OBJECTIVE: To study the method of typing fresh and dry blood.
MATERIALS: Blood Slide, anti sera, titrated anti sera, saline solution, fresh blood, dry
bloodstain, pipettes, test tube and known fresh blood cells.
PROCEDURE:
FRESH BLOOD: Using a lancet or sterilized pin, prick the ring finger of subject whose blood
type is known. Collect two drops of blood in a sterilized test tube containing 3 ml. of saline
solution. Shake the tube gently to produce uniform suspension. Centrifuge the test tube and
discard the clear liquid containing the plasma. Add 3 ml. of saline solution, discard the clear
liquid, and again suspend the cell in saline solution. Place two drops of the suspended cell on the
well of the blood slide and another drop on another well. Study the appearance of the blood cell
under the microscope. Add a drop of anti-A on one well and anti-B of another well. Rotate the
slide slowly for about five minutes and observe which will have agglutination.
DRY BLOOD CELL: Preparation of the stains: Wash the cells of two drops of fresh blood cell
with saline solution in two separate test tube. Suspend the cell in saline solution. Keep the
suspension in the refrigerator.
PREPARATION OF THE ANTI-SERA: Dilute the known anti sera such that each clump
formed when it is treated with blood cells consist of 5 to 7 cells. This procedure is known as
titrating anti-sera.
ABSORPTION: Cut a potion of the stain into smaller pieces and place in two separate test tube.
If the stain is taken on a hard object such as knife etc. scrape the stain with a clean blade and put
two (2) 25 mgs portions in two test tube. To each test tube add saline solution. To each test tube
add a drop of titrated anti-A on one and titrated anti B on the other. Cover the test tube and put in
the refrigerator overnight. Next morning transfer the liquid with anti A mark A and to the test
tube with anti B mark B. To well A drop one drop of cell A suspension. To well B add one drop
of cell 6 suspension. Shake the slide gently for about ten minutes and observe for any
agglutination
OBSERVATION:
1. What is agglutination?
 2. Draw the agglutination observed under the microscope.

3. In grouping fresh blood, why it is necessary to wash the cell before adding the anti-
serum? Explain.

4. Fill in the tables below:

TYPE OF BLOOD
ANTI A
ANTI B

CONCLUSON:
1. Is the clumping of blood cells
2. Copy from the books the possible and impossible children of the parent.
 THE COMPOSITION OF SEMEN
The semen refers to the fluid produced by male sex organ. It The is usually white to yellowish in
color, consisting of two parts the seminal plasma or fluid, and the spermatozoa or sperm cells.
There are usually 70,000,000 to 150.000.000 sperm cells per milliliter of semen. The sperm cells,
as seen under a microscope consist of head, a neck and a tail approximately ten times as long as
the head. During deterioration the bacteria attack first the tail, making identification difficult.
The seminal fluid contains certain substances called flavins, which help give a yellowish color to
semen and cause it to flourish under ultra-violet lights.
THE SEMINAL FLUID: In cases of rape and other sexual crimes, the seminal fluid left by the
attacker on the victim's body or on clothing or furniture presents another powerful source of
evidence. Since the sperm in the fluid remain alive for only a relatively short period, the
condition of the sample can give reasonably accurate indications of the time of the attack. In the
case of secretors, such as sample contains information of blood group and the presence or
absence of other enzymes and proteins that can help to concentrate the search for a potential
suspects nor rather perpetrator's.
Samples of seminal stains or fluid are isolated using tests similar to those used to reveal the
presence of bloodstains. These tests are particularly useful where attempts have been made by
the criminal to remove or wash away incriminating traces. The preceding discussion are intended
to explain what are tests are.
EXAMINATION FOR SEMEN a
a. WET SPECIMEN - Normally, suspected semen may be found undried having the
alkaline odor characteristics for seminal fluid. In this condition examination is relatively
simple. A drop of fluid (of semen) is placed on a glass slide, then a drop of distilled water
is added. A cover slip is placed over the preparation. The specimen is examined under a
high power microscope to determine the presence of sperm cells.
b. DRIED SPECIMEN - Examination most often required is done on dry stain. Physical
and chemical tests are used as preliminary tests. Confirmation is done by microscopic
examination.
1. PHYSICAL EXAMINATION: A general visual examination for grayish-white or
yellowish stain is first made. When dry, semen imparts a starchy stiffness to clothe.
When inspected under ultraviolet light, seminal stain fluoresces. However, this
fluorescence is not specific for semen only but may be observed from other materials or
stains.
2. CHEMICAL EXAMINATION: There are three chemical tests that can be used for
seminal stains. They are the following:
a. Florence Test
b. Barberio's Test; and
c. Acid Phosphatase Test
 The first two are based on the formation of characteristics crystals that are observe under
the microscope. Acid phosphatase is an enzyme found in both animal and plant cells, but
in large concentration inn human semen.

3. MICROSCOPIC EXAMINATION: The only specific test for semen is the

identification of a perm cell under a microscope. There are many factors, which may
affect the detection of sperm cells, making this method difficult. Some of this are nature
of cloth in which the suspected stain is found, age of stain, condition to which stain was
exposed and handling of the specimen.

The presence of sperm cells proves that the stain is semen. However, with the
absence of sperm cells it cannot be concluded that the stain is not of seminal origin.
These are some conditions which may be lead to non detection of semen, like
ASPERMIA, a disorder produced by male organ which produces semen without sperm
cells, and OLIGOSPERMIA, a semen with a very few count of sperm cells.
 SEMINAL STAIN
OBJECTIVE: To Study different test for seminal stain.
MATERIALS: Alleged seminal stain, Florence reagent, glass slide and cover, pipettes, Saline
solution and teasing needles.
PROCEDURES:
A. PRELIMINARY EXAMINATION: Feel the cloth with your finger choose the portion with
a stiff, starchy feeling, and slight deepening of the color. In case there is no deepening or
stiffness, subject the specimen under the ultra violet light. If the cloth is clean and not dark
colored, a bluish fluorescence will be noticed which may indicate the presence of semen.
B. CHEMICAL EXAMINATION: Prepare the Florence reagent by dissolving 1.65 gms. of
iodine crystals in 30 ml. of distilled water Add KI
C. FLORENCE TEST: Cut a portion of the stain and place it on glass slide. Wet the cloth with
three (3) drops of saline solution Tease it carefully and cover with a watch glass. Allow standing
fo 20 minutes. By means of capillary pipettes transfer the liquid to separate slide. Evaporate to
almost dryness at room temperature then add a drop of Florence reagent (freshly prepared).
Examine under the microscope. Observe for the presence of brow elongated choline periodide
crystals and of spermatozoa.

OBSERVATION:
1. Illustrate the appearance of choline periodide crystals.
2. Illustrate the appearance of spermatozoa.

3. State what maybe the result to be able to make a conclusive statement that the stain is
seminal stain