University of Cagayan Valley

BALZAIN HI-WAY, TUGUEGARAO CITY

MODULE NO.3: PRELIM COVERAGE

TITLE SEMEN AND SEMINAL STAINS

INTRODUCTION Semen refers to the body fluid produced by the male sex organ
usually amounting 2.5 to 6.0 ml. seminal fluid per ejaculation.
Each ml contains 100 million or mor spermatozoa. The fresh
ejaculate semen has a gelatinous, sticky, consistency and tends to
become more fluid when exposed. It has two parts, the seminal
fluid and epithelial cells and crystals composed of choline and
lecithin.
The examination of semen and seminal stains is an important
part in the routine investigation of sexual offenses like cases of
rape, adultery, sodony, bestiality and sexual homicide.
LEARNING  Define semen
OBJECTIVES  Identify the parts of the semen
 Discuss the cases wherein ejaculation has no spermatozoa
 Explain the importance of semen and seminal stains in
criminal investigation
 Discuss the four major examinations in conducting semen
and seminal tests
 Describe the mode of collecting, preserving, packaging and
transpiniting semen-stained specimens
SEMEN AND SEMINAL STAIN

The examination of semen and seminal stains is an important part in the routine investigation in
sexual offenses like cases of rape, adultery, sodomy, and sexual homicide.

SEMEN – A whitish fluid of the male reproductive tract consisting of spermatozoa suspended in
secretion of accessory glands.

PARTS OF THE SEMEN

a. Seminal Fluid – has characteristic alkaline odor, it is viscid, gelatinous and sticky.
Becomes more liquid in character when exposed to air for one and half-hour due probably to
enzymatic reaction. Slightly alkaline in reaction. b. Formed Cellular Elements which
includes:

1. Spermatozoa or Sperm Cell – Small objects with a pear-shaped head behind

is a short neck and then a tail of about ten times as long as the head.

2. Epithelial Cells
3. Crystal Of Choline And Lecithin

One point five (1.5) ml to 3.5 ml is the normal quantity of seminal fluid in single
ejaculation 400 to 500 million is the total number of spermatozoa contained in a single
ejaculation from a healthy young man.

CASES WHERE IN EJACULATION HAS NO SPERMATOZOA

1. Males suffering from aspermia.
Aspermia – a condition wherein males have no spermatozoa at all in their seminal fluid.
2. Males suffering from oligospermia.
Oligospermia – a condition whereby males have abnormally low sperm counts or with
few spermatozoa.

These two diseases can be taken from excessive sexual intercourse. Those suffering from chronic
epididymitis and either testicular diseases. Also taken from chronic venereal diseases.
WHERE SEMEN CAN BE FOUND

1. As Fresh
a. Viginal contents of the victim
b. Rectal contents of the victim
2. As Wet or Dried Condition
a. Hair
b. Skin around the genitals
3. As Dry Stains
a. Underclothing

b. Bed clothing

COLLECTION, PRESERVATION, PACKAGING AND TRANSIT SEMEN STAINED

SPICEMENS

1. Seizure of wearing apparel must be done as soon as possible. It often happened that
washing the clothes, chemise, panties and trousers has
destroyed important traces, skirts are the most common parts of wear apparel carrying
seminal stain.

2. In packing of wearing apparel there should be no friction between the apparel and the stain.
The packing of wearing apparel or objects carrying seminal stain must be made in such
manner that there is no friction against the stain. Semen in dried condition is very brittle
and is liable to break into small particles which can be lost. Friction may cause the
breaking of spermatozoa.

3. Specimen should not be rolled for transit. Gently lay between two sheets of cardboard or
similar material which are tied together with a string to avoid friction.

4. Smaller objects like hair should be placed in a test tube and corked. 5. Specimen must be
thoroughly dried before packing. Presence of moisture certain bacteria act on the protein
constituents of semen, digest the dried protein and thus destroy its stiffness.

6. Fluid semen should be placed in a test tube. It may be preserved by a few drops of toulol or
10% solution of formalin during hot weather there is danger of putrefaction.
THE EXAMINATION OF SEMEN AND SEMINAL STAIN.

There are four examinations for seminal stains or seminal fluid in the form of stains namely:

a. Physical Examination b. Microscopic examination c. Chemical

Examination .Biological Examination
c.1. Florence test
b.2. Barberio’s test
c3. Acid Phosphatase test

A. THE PHYSICAL EXAMINATION OF SEMINAL STAIN

a. Semen when dry gives stiff, starchy feeling to the cloth and produces slight deepening of
the color with the disappearance of the odor. Stiffness disappears if specimen is not
properly dried in an open air. Presence of moisture, bacteria will act on the protein
constituent or semen, digest the dried protein thus destroy its stiffness, Also the bacteria
will remove the albunimous matter and also disintegrate the spermatozoa.

b. Seminal stain exhibits bluish fluorescence under the ultraviolet light. Ultraviolet light is
used to locate invisible seminal stain on cloth. It gives bluish fluorescence provided the
cloth is clean and not dark colored. Bluish fluorescence is not specific for seminal stains
and may be seen in some other albunimous materials,

c. Grayish white, sometimes yellowish stain which if it is typical of seminal fluid. d.

Have appearance or outline of contour map.

e. May have a reddish tint in case of old man.

B. THE CHEMICAL EXAMINATION OF SEMINAL STAINS

1. Florence Test – This is known after the name of Dr. Florence of Lysons, who first
introduced it.

Reagent/Chemicals:

Florence reagent (1.65 gram potassium iodide and 2.5 grams iodine in 30 cc of water)

Procedure:
 1. Cut a portion of the stain and divide into small bits then soak in saline solution.

2. Transfer into a slide, tease and evaporate the fluid.

3. Add a drop of Florence reagent and cover with cover slip. 4. Examine
under microscope.

Visible/Positive Result: Crystals of choline periodide, which are dark brown or needle shaped
that occurs singly or in cross or even grouped in clusters. It resembles crystals in shape, size and
color.

Negative reaction maybe due to absence of seminal fluid or spermatic fluid may have not
reacted with the reagent due to the very low choline content because of over dilution. Florence
test is only preliminary, presence of spermatozoa confirms the presence of seminal stain.

Limitation of Florence Test:

1. Clothes with seminal stains are not dried thoroughly so choline periodide is decomposed
completely, so result is negative.

2. If stain is wet and mixed with blood.

3. After 24 hours it is negative due to rapid decomposition but still spermatozoa can still be
detected.

4. Even after a long period (2 ½ years) it will give positive result with Florence test provided
thoroughly dried and preserved and if free from blood and other albuminous substance.

If the seminal stain contains too much albumen as it is mixed with blood, the albumen
interferes to some extent in the test by reacting with so much of the iodine as to leave too
little for the production of Florence’s crystals.

2. Barberio’s Test

Reagent/Chemical
Saturated aqueous or alcoholic solution of picric acid.
 Procedure:

1. Soak a piece of stained material in a 2.5% solution of trichloroacetic acid for one hour in a
test tube.

2. Centrifuge the test tube.

3. Get the liquid part and add to an equal amount of a saturated aqueous or alcoholic solution
of picric acid on a glass slide.

4. Observe under a microscope.

Positive results: Crystal that are slender yellow tinted, rhomboid needles with obtuse angle or
appear as ovoid crystals. These crystals are made of specimen picrate.

Note: Barberio’s test is almost specific for human semen. Seminal stain as old as six years are
said to respond to this test. This test is carried out with fresh, dried or dissolved semen.

3. Acid Phosphate Test – This test is the best way to locate and at the same time
characterized a seminal stain. It has replaced the Florence test in reliability and was
shown to be specific for human and higher apes. The test is based fundamentally upon the
extraordinarily high acid phosphate content of human male ejaculation. Phosphate is the
enzymes present in semen.

Reagent:

Na-a-naphtilphosphate and Fast Blue B Dye

Procedure:
1. Moisen with water a piece of filter paper.
2. Swab the stained area with the filter paper.
3. The acid phosphate will transferred to the filter paper.
4. Add a drop of two sodium alpha-naptylphosphate and Fast Blue B dry. Positive
result: Purple color, Purple color is indicative of acid phosphate.

Alternative Acid-phosphate Test Procedure:

Reagent: 23 grams of sodium chloride, 0.55 ml of glacial acetic acid, 2 grams of sodium acetate
trihydrate in 90 ml water, a suspension of 30 mg of anthraquinone – 1diazonium chloride and 50
grams of calcium – 1 – napthyl phosphate in 1 ml of 1% aerosol.

Procedure;

1. Treat the stained area in a water bath a pH 5 containing

alphanaptylphosphate as a substance and anthraquinone-1-diazonium chloride.

2. Add the above reagents. Positive result: Orange-red pigment Principle of the Test:

Alphanaphtol by the acid phosphate combines with the diazonium salt to form the color. The
reaction takes place for 30 seconds on fresh stains.

Limitation of the Test: Blood lengthens the time but does not interfere.

C. THE MICROSCOPIC EXAMINATION OF SEMEN AND SEMINAL STAIN

The chief purpose of microscopic examination is to determine the presence of spermatozoa. The
identification of spermatozoa is at present the only specific test for semen.

Procedure:

Determination of spermatozoa in fresh semen is relatively easier than in stains.

1. Transfer a drop of specimen to a glass of slide.

2. Add a drop of water or a saline solution and cover with cover slip. 3. Examine
under the high power objective.

4. Observe for the presence of spermatozoa.

 Determination of spermatozoa in seminal stain.
1. A piece of material is teased on a slide in a drop of water. 2. Allow the smear to dry and
then stain with Loffler’s methylene blue for a minute, wash with water, dry and examine
under the microscope.

Limitation of the Microscope Test:

1. Absence of sperm does not prove that the stain have not been produced by human semen.

2. Elements which may obstruct detection of spermatozoa:

a. Nature of fabric
b. Age of stain
c. Condition to which the stain was exposed before reaching the laboratory.

d. Handling the specimen

3. Some medical jurist believes that these can be no semen without the presence of
spermatozoa, but not tru in case of aspermia or oligospermia.

D. BIOLOGICAL EXAMINATION OF SEMEN AND SEMINAL STAIN

The spermato-precipitins are of value in the identification of seminal fluid in certain cases
like for example, bestiality when it may be desirable to differentiate between the human
seminal fluid from that of animal.

This test was originally proposed by Farnum in 1901. He injected human semen to a
rabbit from five to eight times of intervals from six to eight days. The serum obtained from
the blood of the rabbit give a precipitate with both recent and old emulsions of human semen.
In 1928, Hectoem and Rustinant showed that an antiserum produced by immunizing rabbits
with human semen is both specie specific and semen specific i.e.; it gives a positive reaction
with human blood.

Limitation of the Test:

The bacterial action that produces disintegration of the spermatozoa in seminal stain is equally
effective in decomposing and digesting the protein constituent of semen that acts the antigen
producing antibodies. Such seminal stains with their protein constituents completely
disintegrated cannot possibly give a positive precipitin reaction.

OTHER STAINS OF MEDICO-LEGAL INTEREST

1. Obstetrical and gynecological stains. Examination at the scene of the crime in cases of
criminal abortion, infacticide and sex offenses may lead to the discovery of bed linen,
towels, chemise, skirts, mattresses, blankets etc. which have stains

2. Excrements:

Adults – yellowish brown

Infant – greenish yellow
3. Paints stains: The criminal, in committing a crime may have brushed against a newly
painted wall or wall with loose water cement paint and may therefore carry some of the
paint of his clothing.

4. Rust stains: Resembles blood stains.

Rust – reddish-brown in color, insoluble in water and soluble in dilute acid.

5. Synthetic dyes: Resembles old blood stains but can be recognized by treating with strong
acids and alkaline.

6. Mineral stains. These are due to red paints containing oxides of iron. 7. Stains of vegetable
origin: Stains resembling blood may be produced by fruit juices like mulberry, mangosteen.

Almost all the above can be differentiated from bloodstains by actions of chemicals. The
above give reaction while blood does not.

Prepared by:

NOEL S. PENETRANTE
Forensic Chemistry Teacher