Seminal fluid is a complex mixture of secretions from at least four male urogenital glands. The seminal vesicle gland contributes approximately 60% to this mixture, the prostate gland contributes approximately 30%, and the combined contribution of the epididymis and bulbourethral glands account for the remaining 10% Confirmatory Tests for semen: 1- The Christmas Tree Stain: The most reliable confirmation for the presence of semen is the positive visual identification of sperm cells (or spermatozoa) using the Christmas tree stain.

1. Confirmatory Test For Semen Identification
Presented by:
Parth Chauhan
LNJN NICFS, MHA, Delhi

2. About Semen About Tests
• It is a thick, yellowish to off white, alkaline
secretion having a characteristic odour.
• pH of around 7.5 – 8 (slightly alkaline)
• Typical ejaculation – 2 ml to 5 ml of semen
• Major content of seminal fluid consist of :
A. Fructose
B. water
C. Iron
D. Choline
E. Spermine
F. Seminal Acid Phosphatase (SAP)
J. LDH (Lactose Dehydrogenase enzyme)
K. Prostate Specific Antigen (PSA)
• Confirmatory Tests of semen includes:
A. Microscopic Examination of Spermatozoa
(i) Examination through Gram Staining
(ii) Hematoxylene & Eosine Staining
(iii) Christmas Tress Staining
B. Anti - Prostate Specific Antigen (PSA) Test

3. Gram staining of Spermatozoa
1. Slide preparation for
staining :
1cm x 1cm portion
Acidulated water in test tube
Vortex (freeing spermatozoa
from Dried stain)
Centrifuge (30 sec.)
Withdraw Supernatant
placing insoluble on clean
slide
Fixing with H2SO4 acid and
dry
2. Gram Staining :
Reagent Preparation :
Reagent 1: Ammonium oxalate crystal violet solution
A: Add 0.2 g of crystal violet dye to 20 ml of 95% ethanol.
B: Add 0.8 g of Ammonium oxalate to 80 ml of distilled water.
Reagent 2: Gram’s iodine
Iodine 1 g
Potassium Iodide 2 g
Distilled water 300 ml
Reagent 3: Decolorizer – 95% ethanol
Reagent 4: Safranin solution
Safranin (2.5% in 95% ethanol) 10 ml
Distilled water 300 ml

4. 2. Gram Staining :
Procedure :
Fixing smear by gentle heating
Add reagent 1(Crystal Violet Solution) for one minute
Rinse with tap water
Add reagent 2 (Gram Iodine Solution) for one minute
Rinse with tap water
Add reagent 3 (Decolorizer) for one minute
Rinse with tap water
Add reagent 4 (Safranin Solution)
Air dry and examine in oil immersion at 1000X
3. Observations :
Spermatozoa will appear as differentially stained
purple bodies
4. Grading of smear :
+ Few; difficult to locate
++ Some in some fields
+++ Some in many fields; easy to locate
++++ Many in most fields

5. Fig 1: Spermatozoa will appear as differentially stained purple bodies, somewhat
oval in shape with a clearly discernible acrosomal cap

6. Hematoxylene-Eosin (H&E) Staining
1. Slide preparation for
staining :
1cm x 1cm portion
Acidulated water in test tube
Vortex (freeing spermatozoa
from Dried stain)
Centrifuge (30 sec.)
Withdraw Supernatant
placing insoluble on clean
slide
Fixing with H2SO4 acid and
dry
2. H&E Staining :
Reagent Preparation :
Reagent 1: Hematoxylene stain:
Haematein 0.4 g
Potassium alum 5 g
Glycerine 30 ml
Distilled water 70 ml
Reagent 2: Eosin Stain
Eosin 1 g
Distilled water 100 ml

7. 2. H&E Staining :
Procedure :
Fixing smear by gentle heating
Add reagent 1(Hematoxylene stain) for one minute
Rinse with tap water
Add reagent 2 (Eosin stain) for one minute
Rinse with tap water
Allow slide to dry on hot plate
place cover slip over the top of stain
Examine with 40X objective
3. Observations :
Spermatozoa stained with dark purple nucleus
with red cytoplasm.
4. Grading of smear :
+ Few; difficult to locate
++ Some in some fields
+++ Some in many fields; easy to locate
++++ Many in most fields

8. Fig 2: Spermatozoa stained with H-E Stain

9. Christmas Tree Staining
1. Slide preparation for
staining :
1cm x 1cm portion
Acidulated water in test tube
Vortex (freeing spermatozoa
from Dried stain)
Centrifuge (30 sec.)
Withdraw Supernatant
placing insoluble on clean
slide
Fixing with H2SO4 acid and
dry
2. Christmas Tree Staining :
Reagent Preparation :
Reagent 1: Karnechtrot Reagent
A: Add 5 g of Aluminium sulphate in 100 ml of hot water.
B: Add 0.1 g nuclear fast red dye. Cool and filter
Reagent 2: Picroindigocarmine Reagent
A: Add 4 g of Picric acid to 300 ml of distilled water. Stand
all night
B: Dissolve 1 g of indigocarmine dye in the solution and filter

10. 2. Christmas Tree Staining :
Procedure :
Fixing smear by gentle heating
Add reagent 1(Karnechtrot reagent) for 5 minute
Rinse with tap water
Add reagent 2 (Picroindigocarmine Reagent
) for 20 seconds.
Rinse with ethanol
Allow slide to dry on hot plate
place cover slip over the top of stain
Used immersion oil to view in 1000X
3. Observations :
Tip of head – pink
Bottom of head – dark red
Middle piece – blue
Tail – yellowish green
4. Grading of smear :
+ Few; difficult to locate
++ Some in some fields
+++ Some in many fields; easy to locate
++++ Many in most fields

11. Fig 3: Spermatozoa stained with Christmas tree stain

12. About Prostate Specific Antigen (PSA)
• Also known as Kallikrein-3 (KLK3) or
P30 Antigen.
• Molecular weight of PSA is 30kDa so
called P30 Antigen.
• It is a member of Kallikrein Family and is
encoded by KLK3 locus on chromosome
19 so named Kallikrein-3.
• Also it is responsible for hydrolyzing
Seminogelin (Sg) which helps in gel
formation in semen.
There are different methods to perform PSA test :
1. Ouchterlony Double Diffusion
2. Immunochromatographic assay (Semtec / ABAcard )
3. ELISA
4. Crossover electrophoresis
Advantages of PSA over other techniques:
1. Detection of seminal stains in Oligospermic or
Aspermic cases.
FLUID PSA(ng/ml)
Semen 200,000-5.5 million
Amniotic fluid 0.60-8.98
Breast milk 0.47-100
Saliva 0
Female Urine 0.12-3.72

13. • Agarose plate is prepared with two
opposite wells
• Extract of suspected stain is placed at
cathode well.
• Anti p30 serum is placed loaded at anode
well.
• Electrophoresis is carried out at 120V for
20 minutes.
• Observed result against light shows fine
precipitate line between the two wells.
• Recheck the step by staining the plate with
Amido black for 10 minutes which shows
deep blue/black bands.
3. Observations :
The precipitate line at the center of the plate
shows the presence of antigen-antibody reaction
which confirms the presence of seminal fluid.

14. Reference:
• Li, R. (2013). Forensic biology: Identification and DNA analysis of biological
evidence.
• Balk SP, Ko YJ, Bubley GJ (January 2003). "Biology of prostate-specific antigen". Journal of
Clinical Oncology. 21 (2): 383–91. doi:10.1200/JCO.2003.02.083. PMID 12525533.
• DFSS Biology Manual

15. Thank You