This ppt basically on stains such as seminal, saliva

1. STAINS DR GURJEET SINGH
PGT
AMCH

2.  Introduction
 Morphology
 Composition
 Examination
 Medico legal significance

3. INTRODUCTION
 Semen also known as seminal fluid.
 Its an organic fluid it contain spermatozoa.
 It is secreted by the gonads (sexual glands) male organs.

4. Spermatozoa are
produced in the
seminiferous tubules
within the testes.
Epididymis is the first
section of the sex
accessory ducts from
which spermatozoa travel
first time outside the
body.
It is later merge with vas
deferens
Than its passes through
sex accessory glands.

5. Diagram showing testes, sex accessory duct, and sex accessory
glands

6. Why need seminal stains
 Objective :- Seminal Stains have to be detected / examined in criminal and
civil cases.
Criminal Cases
o Rape / Attempted rape
o Sodomy
o Bestiality
o Sexual homicide of female
Civil Cases
o Disputed Paternity
o Legitimacy
o Artificial Insemination
o Compensation on
grounds of acquires
sterility.
o Divorce

7. Collections
 Clothing
 Vaginal Fluid.
 Dried stains on other parts of body.
 Matted pubic hair
 Stains on smooth surface :- gently scraped in glass container and
preserved.

8. Methods
Physical
Chemical
Microscopic
Electrophoretic

9. SCREENING TESTS
 Physical Examination:-
Fresh semen is a whitish or
yellowish – white in colour,
slightly viscous, jelly like, sticky
and has a characteristic odour.
o Dried seminal stains on clothes are
greyish- white or yellowish - grey in
colour, show an irregular outline and
starchy hard in feeling.
o Its shows bluish – white colour under
UV light

10. Diagram showing Seminal stain under the UV
Light

11. Presumptive tests
 Florence test:- A drop of watery solution of suspected stain place on slide
dry it covered with a cover slip and a drop of Florence solution ( 8 % of
iodine in water containing 5% of potassium iodide)
 If semen is present, dark brown rhombic crystals resembling hemin if
seminal matter present.
False Negative
Low choline
Decomposed stain
False positive
Other fluid which contain
choline.

14. Barberio’s Test
 This test depends on the presence of spermine.
 Its also positive without the presence of spermatozoa.

15. Acid Phosphatase Test
α- naphthyl acid phosphate Sodium phosphate + naphthol
Acid phosphatase
Naphthol + Brentamine Purple dark colour

16. Other Tests
 Creatine phosphokinase test-: >400 units/ml
 Choline Spermine test
 Ammonium molybdate test

17. Microscopic Test
Presence of
spermatozoa under
light microscope is a
confirmatory
method.
Confirmatory
testing involves
solubilization of
sample followed
by centrifugation.
Which yield a
supernatant and a
cell pellet.
Cell pellet is used
to detect
spermatozoa and
DNA analysis.
Supernatant is
used when sperm
are not detected,
for genetic
profiling.

18.  Contaminated sample with saliva, vaginal secretions in which selective
degradation to be done by treating the cell extract with a mixture of
proteinase K and sodium dodecyl sulfate.

19. Morphology of spermatozoa
Size of Human Spermatozoa is 55
µ in length head is ovoid and
flattened,5 x 3.5 in dimension.
It has a short head and a long
filamentous tail 50 µ .

20. Procedure
A small piece of stained
fabric is moistened with a
few drops of 1% HCL in a
watch glass.
Slides are prepared by
dabbing the fabric gently
on them , dried and then
stained.

21. Slide is stained either
with haematoxylin for 2 –
5 min and eosin for 3
min.
Posterior half to one third
of head and the tail takes
eosin and is stained deep
red or pink.
Anterior half to two third
takes very light, faint
basic or blue stain.

22. Christmas tree stain
This staining technique is consist of
nuclear fast red ( red stain for sperm red)
picroindigocarmine ( green – counter –
stain for the tail and other cytoplasmic
material) and are sometimes referred to
as Christmas tree.

23. Other Stains
Papanicolaou Staining :- In
pap smear , acrosome is
stained pink, post acrosome
is dark blue, tail pink.
Baecchi Stain:- uses acid
fuchsin and methyl blue,
sperms heads stain red and
nuclei blue.

24. Spermatozoa under light microscope

25. At body
temperature
sperms retains
full motility in
vagina between
6 – 12 hours.
The sperm
remain motile
in the uterine
cavity 3 – 7
days.
Non motile
sperm may be
seen in oral
cavity 2 – 31
hours.
4- 113 hours in
rectum, 2 – 44
hours in anus.

26. Non-cellular semen markers
Acid phosphatase Test :- 340 – 360 Bodansky
units/ml.
Prostate specific antigen or PSA (P30):- can be
detected in dried and old stains even after 10
years also in cadaver.
ABA card test strips is accepted method for
identification of semen.

27. Electrophoretic test
 Lactate dehydrogenase :- the sperm specific isoenzyme LDHx stable in
stain for four week.
 This iso-enzyme pattern of human semen is different from that of common
animals.
 Positive results obtained in much larger number than is possible with
microscopic examination.

28. SALIVA
Identification of saliva on bite
marks, cigarette secretor or bidi
ends and on clothes, and
determination of secretor status
is important in criminal offences.
Its depends on presence of
enzyme α – amylase and buccal
epithelial cells

29.  Rapid diffusion utilize agar gel containing starch, the α – amylase activity
is detected by the classical starch iodine that give a characteristic purple
reaction.

30. PHADEBAS TEST
 Starch is covalently linked to a dye such as Cibacron blue or procion red to
form insoluble complex.
 Subsequently to α – amylase activity the dye is released from the complex
and become soluble and change in colour can be measured by
spectrophotometry.

32. URINE
 Main components :-
i) UREA
ii) Creatinine
Presumptive test – urine
 Jaffe test – Based on detection of creatinine.
 Suspected urine sample + picric acid + 5 NaOH immediate orange
colour.

33. Fecal Matter
 Human waste has bilirubin.
 Presumptive test – Edelman’s reagent
 Faecal stain Bilirubin urobilinogen Urobilin
Shine UV light Edelman’s reagent Green Fluorescence

34. Sweat
 Suspected Sweat sample + Crystals violet deep purple colour

35. MEDICO –LEGAL ASPECTS
 Disputed Paternity:- The question of disputed paternity arise in the court
in the following conditions:
 Divorce
 legitimacy
 Maintenance claims
 Share of property

36. Identification of
victim or
offenders.
Stains due to
body fluids : The
blood group
antigens can be
demonstrated in
stains on clothes
due to semen,
sweat or saliva
(secretors)
Crime scene
reconstruction
Malingering Cause of death Time since death

37. References
1) Saukko P, Knight B. Knight’s forensic
pathology.4th Ed. CRC Press; 2015
2)DiMaio VJM, Kimberley Molina D. Di Maio’s
Forensic Pathology. Boca Raton: CRC Press;
3)Subrahmanyam BV. Parikh’s textbook of
jurisprudence, forensic medicine and
classrooms and courtrooms. 8th ed. New Delhi,
India: CBS Publishers & Distributors; 2019.
4)Reddy KSN, Reddy M. The synopsis of
medicine & toxicology the synopsis of forensic
medicine & toxicology. New Delhi, India:
Brothers Medical; 2022.