Examination of Semen in Forensic Science

1. Locating Seminal Stains

Visual Inspection

Seminal stains are initially identified through visual examination of clothing, bedding, furniture, or other surfaces. Semen stains often appear as crusty or stiff patches on fabric.

Alternate Light Source (ALS)

An Alternate Light Source (ALS), such as UV light or blue light with orange filters, is commonly used to detect biological stains. Semen exhibits fluorescence under certain wavelengths, making the stains easier to locate.

Preliminary Examination of Semen

1. Brentamine Fast Blue Test / Colorimetric Acid Phosphatase (AP) Assay

Principle

Acid phosphatase present in semen hydrolyzes α-naphthyl phosphate to α-naphthol. The α-naphthol then reacts with Brentamine Fast Blue reagent to produce a purple azo dye.

Procedure

1. Take the swab or stain sample on filter paper.

2. Add α-naphthyl phosphate reagent.

3. Add Fast Blue B reagent to the sample.

Interpretation

• Positive Result: Rapid purple or violet color develops within 30 seconds.

• Negative Result: No color change or delayed color development is observed.

2. Barberio Test

Principle

The Barberio test detects spermine, a compound responsible for the characteristic odor of semen.

Procedure

1. Add saturated picric acid solution to the suspected sample.

2. Observe for crystal formation under the microscope.

Interpretation

Formation of yellow needle-shaped crystals confirms the presence of spermine.

Limitations

This test is less commonly used because of its lower specificity.

3. Florence Test

Principle

The Florence test detects choline, an important component of semen.

Procedure

1. Add Florence reagent (potassium iodide and iodine solution) to the sample.

2. Observe the reaction microscopically.

Interpretation

Formation of brown rhombic crystals of choline periodide indicates a positive result.

Limitations

Choline may also be present in other body fluids such as sweat, reducing the specificity of the test.

Confirmatory Tests for Semen

1. Christmas Tree Stain (Kernechtrot–Picroindigocarmine Stain)

Principle

This staining method differentially stains various components of sperm cells for microscopic identification.

Procedure

1. Prepare a slide containing the semen stain sample.

2. Apply Nuclear Fast Red (NFR) stain followed by Picroindigocarmine (PIC) stain.

3. Examine the stained slide under a microscope.

Observation

• Sperm heads stain red.

• Sperm tails stain green.

Microscopy

The slide is examined under 400× or 1000× magnification for sperm identification.

2. Prostate-Specific Antigen (PSA or p30) Test

Principle

Prostate-Specific Antigen (PSA) is a protein secreted by the prostate gland and is abundant in seminal plasma. Detection of PSA confirms the presence of semen.

Procedure

1. Use immunochromatographic or ELISA-based methods.

2. Extract the suspected stain sample in a suitable buffer solution.

3. Apply the extract onto the test strip.

4. Antibodies present on the strip bind with PSA to produce a visible test line.

3. RSID-Semen Test

Principle

The RSID-Semen test detects semenogelin, a protein secreted by the seminal vesicles.

Procedure

1. Perform the rapid immunochromatographic test according to standard protocol.

2. Observe the development of the test line.

Interpretation

A positive result confirms the presence of semen in the sample.

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