PRACTICAL NO, 4

Aim: Immune-double ditfusion method: Detemination of species from blood stains.

Materials Required: Agar Powder, Conical Flask, Distilled Water, Oven, Petri Dish, Blood Stain
Sample, micropipette, Dropper, Weighing Machine, NaCI.
Theory:
In Forensic Science Laboratories if evidence is identified as blood then the next analysis to be
done is identification of species. If the blood sample is of non-human origin so, no further analysis
is roquied. For identification of species immune-double diffusion method is used which is also
known as Ouchterlony double immuno-diffusion. This method is based on antibody antigen
reaction. In this method precipitin line is formed which is indication of positive reaction between
antibody and antigen.
Procedure:
(a) Preparation of Agar Gel plate
Weigh 0.3gm agar powder and put it in conical flask.
Then add 30mldistilled water and stir it well.
Put the flask in oven for 1-2 min to dissolve it and it will become a clear solution.
After that take a clean Petri dish and pour the solution in it and leave it for 10-15 min to
solidify it.
Punch well in it with the help of dropper,each about Smm apart.
(b) Preparation of sample
Take blood stain sample and cut a portion of it and put it in normal saline.
..
Afler 30mins solution become light brown in colour.
(c) Loading of prepared sample and antisera
With the help of micropipette load sample in one well and anti-H(Human) antisera in its
adjacent well.
Now cover the petridish with other petridish and put in a box having wet blotting paper at
bottom.
Leave it overnight at cool place.
Observe precipitin line next day.

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Observations:A white precipitin line is observed between the wells.

Precipitin line showing positive reaction between antibody and antigen

Result: According to above observation, it may be opined that the sample has human origin.
Precautions:
1. Weighing of agar powder should be done properly.
2. Formation of well should be done carefully.
3. Sample should not overflow from well.

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 PRACTICAL NO. 5

Aim:-Spectroscopic analysis of blood. Mercaptoethanol, Ammonia

stain sample, Sodium lauryl sulphate,
Material Required:- Blood
Spectrophotomee:
Solution, Distilled water, breaker, measuring cylinder,
found evidence at scene of crime and can be analysed by different
Principle:- Blood is frequently based on oxidation of
the method is spectroscopic analysis. This method is
ctiods, one of
oxyhaemoglobin is compared with standards or
haemoglobin and then maximum absorption of
control samples.
Preparation of reagents:
Solution 1: 0.2% Sodium lauryl sulphate in water.
Solution 2: 0.2% Mercaptoethanol in 19% NH3 solution.
Procedure:
and put it in 10 ml of Solution 1.
Take lcm long stained thread of sample
2 Incubate it at 370C for 15-20 minutes.

3. Now add 10ml of Solution 2 and mix it.

4. Transfer liquid to a cuvette.

at 560 mm against a reaction blank until
5. On a Spectrophotometer, monitor the reaction
absorption reaches maximum and set it to zero.
between 600 and 500
6. When the reaction is complete, after 5-10minutes, scan the sample
nm.

7 And record number of peaks and maximum absorption.

Observations:- Two peaks, which are clearly defined at 558 nm and 529 nm.

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absoance
Relatve Oxyhemoglobin

10

Deoxyhemog'obin

620 640 660

480 500 520 640 560 580 600
Wavelength (nm)

Results: It is opined that the given sample may contain blood.

Precautions:
1. Blank doesn't contain sample.
2. Absorption should be set at zero by the help of blank.

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