FORENSIC CHEMISTRY AND TOXICOLOGY

Chapter 3: Semen and Seminal Stains

Learning Outcomes
At the end of this chapter, the students will be able to:

1. Identify the physical and chemical properties of semen and seminal fluid.
2. Discuss the various tests performed for semen analysis.
3. Understand the techniques for semen examination.
4. Analyse various types of sexual assault cases.

SEMEN AND SEMINAL STAIN

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

SEMEN – A whitish fluid of the male reproductive tract consisting of spermatozoa

suspended in the secretion of accessory glands.

PARTS OF THE SEMEN

a. Seminal Fluid – has characteristic alkaline odor, 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 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 ejaculate from a healthy young man.

CASES WHEREIN EJACULATION HAS NO SPERMATOZOA

1. Aspermia – a condition wherein males have no spermatozoa at all in their
seminal fluid.
2. 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 other testicular diseases. Also taken from
venereal diseases.

LOCATION OF SEMEN AND SEMEN STAIN AS EVIDENCE

1. As Fresh
a. Vaginal 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 OF SPECIMEN

1. Seizure of apparel must be done as soon as possible.
2. In packaging of wearing apparel there should be no friction between the apparel and
the stain.
3. Specimen should not be rolled for transit.
4. Smaller objects like hair should be placed in a test tube and corked.
5. Specimen should be thoroughly dried before packing.
6. Fluid semen should be placed in a test tube. It may be preserved by a few drops of
10% solution of formalin during hot weather.

SEMEN AND SEMINAL STAINS EXAMINATION

There are four examinations for seminal stains or seminal fluid in the form of
stains namely:
1. Physical Examination
 2. Chemical Examination
a. Florence Test
b. Barbeiro’s Test
c. Acid-phosphates test
3. Microscopic Examination
4. Biological Examination

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 open air. Presence of
moisture, bacteria will act on the protein constituent or semen, digest the
dried protein thus destroy its stiffness. Also, bacteria will remove the
albuminous matter and also disintegrate the spermatozoa.

b. Seminal stains exhibit 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 albuminous materials.

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

d. Have appearance or outline of contour map
e. May have a reddish tint in case of old man

*C. DETERMINATION OF SPERMATOZOA IN FRESH SEMEN:

1. Transfer a drop of specimen to a glass slide

2. Add a drop of water or saline solution and cover with cover slip
3. Examine under the microscope
4. Observe for the presence of spermatozoa

ELEMENTS WHICH MAY OBSTRUCT DETECTION OF SPERMATOZOA

1. Nature of fabric
2. Age of stain
3. Condition to which the stain was exposed reaching the laboratory
4. Handling of the specimen

*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 an animal.
 This test was originally proposed by Farnum in 1901.

OTHER STAINS OF MEDICO-LEGAL INTEREST

1. Obstetrical and gynecological stains: Examination at the scene of the crime in cases
of criminal abortion, infanticide 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 on the 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 bloodstains 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 produced by fruit juices like
mulberry, mangosteen.

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

B. THE CHEMICAL EXAMINATION OF SEMINAL STAINS

1. Florence test – 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. Adda drop of Florence reagent and cover with cover slip.
4. Examine under the microscope.
Visible/ Positive result: Crystals of choline periodide, which are dark brown, rhombic
or needle shaped that occur singly or in cross or even grouped in clusters. It resembles
haemin crystals in shape, size and color.

Negative reaction maybe due to absence of seminal fluid or spermatic fluid may
have not reacted due 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 the stain is wet and mixed with blood.
3. After 24 hours it is negative due to rapid decomposition but still spermatozoa can be
detected.
4. Even after the 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 albumin as it is mixed with blood, the albumin
interferes to some extent in the test by reacting with so much of the iodine 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 1 hour in
a test tube.
2. Centrifuge the test tube.
3. Get the clear 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 Phosphatase Test – This test is the best way to locate and 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 phosphatase content of human male ejaculate.
Phosphatase is the enzymes present in semen.

Reagent:
Na-a-naphthyl phosphate and Fast Blue B dye

Procedure:
1. Moisten with water a piece of filter paper.
2. Swab the stained area with the filter paper.
3. The acid phosphatase will be transferred to the filter paper.
4. Add a drop or two of sodium alpha-naphthyl phosphate and Fast Blue B dye.

Positive Result: Purple color is indicative of acid phosphatase.

Alternative Acid-phosphatase 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-1-diazonium
chloride and 50 grams of calcium – 1 – naphthyl phosphate in 1 ml of 1% aerosol.

Procedure:
1. Treat the stained area in a water bath a pH 5 containing alpha-naphthyl phosphate as
a substance and anthraquinone -1-diazonium chloride.
2. Add the above reagents.

Positive result: Orange-red pigment

Principle of the Test:

Alpha naphthol by the acid phosphatase 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 interefere.