Preliminary tests for Semen

Preliminary tests for the detection of semen stains are crucial in forensic investigations, as they allow investigators to quickly and accurately identify potential evidence at a crime scene. In this chapter, we will discuss some of the most commonly used preliminary tests for detecting semen stains:

PHYSICAL EXAMINATION

1. Colour, Texture, and Appearance

   Seminal stains, also known as semen stains, typically appear as a white or yellowish, sticky, and cloudy fluid to the naked eye. They may also have a slightly opaque or translucent appearance which can vary depending on the amount of semen present, the age of the stain, and environmental conditions. For example, a fresh stain may appear more liquid and have a brighter colour, whereas an older stain may have dried out, becoming stiffer and darker in colour. Additionally, if the stain has been exposed to heat, light or other environmental conditions, it may have discoloured or become less visible.

   It's important to note that seminal stains can be hard to see with the naked eye, especially if they are small or if the surface they are on is already dirty or discoloured. This is why forensic experts often use specialized lighting techniques for visualization.

2. Fluorescence

   Visual examination of semen stains is achieved using the phenomenon of fluorescence, where the semen stain emits light at specific wavelengths when exposed to certain light sources. Seminal stains can be detected using alternative lighting techniques like ultraviolet (UV) light. UV light makes semen fluorescence, appearing as a light blue or white glow. The excitation wavelengths used for the detection of seminal stains typically fall between 300-500nm and the emission ranges between 400 and 700nm. The emitted light can help forensic experts locate and identify the presence of semen stains on various surfaces. However, this technique is not specific to semen alone and other bodily fluids such as saliva may also exhibit similar fluorescence, usually at a lower intensity.

CHEMICAL EXAMINATION

1. FLORENCE TEST

Figure: Florence Test for the detection of semen

Principle: Detection of choline secreted by the prostate gland.

Preparation of Florence Reagent: Add 1.65g of potassium iodide (KI), 2.54g of iodine, 30ml distilled water and mix well.

Procedure:

1. Take 1-2 drops of aqueous extract of the stain on a slide.

2. Add a drop of Florence reagent from the sides and allow it to mix slowly under a cover slip.

3. Observe under a microscope at 10X and 40X magnification. The presence of dark brown rhombic crystals of choline periodide indicates the presence of semen in Florence test.

2. BARBERIO’S TEST

Figure: Barberio’s Test for the detection of semen

Principle: Detection of spermine secreted by the prostate gland.

Preparation of Barberio’s Reagent: Add 1g of picric acid in 100mL concentrated hydrochloric acid (HCl) and mix well. It is important to note that picric acid and concentrated HCl are hazardous chemicals and should be handled using protective equipment, such as gloves and safety glasses.

Procedure:

1. Take 1-2 drops of aqueous extract of the stain on a slide.

2. Add a drop of Barberio’s reagent from the sides and allow it to mix slowly under a cover slip.

3. Observe under a microscope at 10X and 40X magnification. The presence of yellow-coloured needle-shaped crystals of spermine picrate indicated the presence of semen in Barberio’s test.

3. ACID PHOSPHATASE TEST

Figure: Acid Phosphatase test for the detection of semen

Principle: Detection of acid phosphatase secreted by the prostate gland.

Preparation of Reagents:

a) Buffer: Add 1ml Glacial acetic acid and 2g of Sodium acetate anhydrous to 100ml of distilled water. The buffer is used to maintain a stable pH environment for the reaction.

b) α-naphthyl phosphate solution: To 50ml buffer, add 126mg α-naphthyl phosphate and mix well.

c) Napthanil diazo Blue B solution: To 50ml buffer, add 250mg napthanil diazo Blue B (also known as Fast Blue B) and mix well.

Procedure:

1. Obtain a sample of the suspected semen stain. This can be collected from various surfaces, including clothing, skin, etc. Since prostate acid phosphatase is water soluble, a moistened cotton swab or piece of filter paper can be used to transfer a small amount of sample from a stain by briefly pressing it onto the questioned stain area.

2. Add 1-2 drops of α-naphthyl phosphate solution to the suspected stain, followed by 1-2 drops of napthanil diazo Blue B solution.

3. Observe the sample for a colour change. A purple colouration developed within 1 minute is considered a positive indication for semen. Colour that develops after more than 1 minute may arise from the activity of non-prostatic acid phosphatase. The intensity of the colour can be used to determine the amount of acid phosphatase present in the sample.

‼️ False Positive Results

It is important to note that the acid phosphatase test is not 100% specific for semen, and it may give false positive results with other bodily fluids that contain acid phosphatase such as blood, urine, and vaginal secretions. Therefore, it is important to confirm the results of preliminary tests, including the acid phosphatase test, with a more specific confirmatory test.

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