Preliminary and Confirmatory Test for Semen


Semen is a bodily fluid secreted by males for the purpose of reproduction. Semen may be divided into two components for forensic purposes: seminal fluid and spermatozoa. Seminal fluid is a protein-rich bodily fluid that is produced primarily by the prostate and seminal vesicles.

A healthy man typically secrete 2 to 6 ml of semen, containing 100 to 150 million sperm cells per milliliter. It has a thick, yellowish white, opalescent appearance and secretions with the distinctive seminal odour.

Spermatozoa, sometimes known as “sperm,” are male gametes or sex cells generated in the testis. Not all males are able to produce spermatozoa. Seminal fluid in males who have undergone a vasectomy, certain birth abnormalities, or as a result of certain disorders will either be devoid of or contain extremely few spermatozoa. These are known as Oligospermins (low sperm count) and Azoospermins (no sperm).

Presumptive Tests

● Alternative Light Sources (ALS)

Semen will glow under certain lighting because it contains compounds like flavin and choline-conjugated proteins. Depending on the lighting system used, its colour can vary from blue to yellow. This detection method is extremely speculative since numerous chemicals, both natural and synthetic, glow similarly to semen.

● Seminal Acid Phosphatase Test (SAP)

Seminal Acid Phosphatase Test (SAP) Also referred to as the Walker Test or Brentamine spot test. A dark purple hue will be produced by AP in less than a minute when it is combined with Brentamine Fast Blue and Alpha-Naphthyl Acid Phosphate.

● Prostate Specific Antigen

Prostate Specific Antigen Tests find prostate specific antigen (PSA). Male prostate glands produce significant levels of PSA. In extremely minute quantities, this antigen can also be discovered in perspiration and faeces. According to studies, PSA can also be found in breast milk and female urine. When interpreting PSA readings that are positive but aren’t verified by sperm presence, caution is advised.

(This assessment is no longer valid.)

● Choline Test

A drop of Florence iodine is added after the stain extract is placed on a microscope slide. After setting the cover slip, wait 10 minutes. Choline per iodide crystals will develop in a brown colour, indicating the presence of semen in the stain.

● Spermin Test

To prepare the extract, centrifuge the stained cloth for an hour after soaking it in a 2.5 percent solution of trichloroacetic acid in a test tube. On a microscope slide, add an equal volume of picric acid aqueous solution along with the supernatant. Spermin picrate will create yellow-colored, obtuse or rhombic prism-shaped crystals that are favourable indicators of the presence of human sperm.

Confirmatory Tests

● Christmas Tree Stain

Sperm cells that can be positively identified visually using a stain. In order to create this characteristic stain, two major reagents are applied sequentially: Nuclear Fast Red provides the sperm heads a read colour and the tip of the heads a pink colour, while sperm neck and tail sections are stained green and blue by Picroindigocarmine.

● RSID (Rapid Stain Identification) Test for Semen

It indicates the presence of semonogelin, or a seminal vesicle-specific antigen. Because this antigen is specific to human sperm, there is no cross reactivity with other body fluids in males and females, or with sperm from other species. This test can also detect sperm even if the stain was kept in less-than-ideal conditions.

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