ACKNOWLEDGEMENT

Perhaps nothing in the world can be achieved in isolation. Therefore we need co-operation of other for that training report is an Outcome of constructive support and co-operation of many individual. First of all I would like to express myself I am pursuing M.Sc. BIOTECNOLOGY FROM PATNA UNIVERSITY. I would like to express my deepest gratitude and independent to my honourable faculty and guide. My special thanks to Mr. U.K.Sinha (director) and Mr. Shiv Kr. Tiwari (Serology) state forensic laboratory Patna for allowing me to do training in this (State Forensic Science Laboratory) reputed organization more over methodology has helped me in refining my study to make it more effective. I would like to thank specially Mr Partha Banerjee (Senior Scientific Officer) for guiding me throughout my project in the highly skillful manner.My sincere thanks to MISS Ambalika Tripathi (Senior Scientific Officer), Neeraj Varshney (Senior Scientific Officer), for guiding me in narcotics division. I would also like to thanks Mr. Das Sir and R.K. Sinha (BIOLOGY) and Mr Ajay Kumar(SEROLOGY) and Dr. Rekha Kumari. Toxicology Division responded on me and provided me valuable information in technical or practical work. I express my hearties to my parents, family membersand all other friends who directly helped me. Last but not the least I am thank full to all mighty for showering their blessing on me.

INTRODUCTION Forensic science (often shortened to forensics) is the application of a broad spectrum of sciences to answer questions of interest to a legal system. This may be in relation to a crime or a civil action. The word forensic comes from the Latin adjective forensis, meaning "of or before the forum." In Roman times, a criminal charge meant presenting the case before a group of public individuals in the forum. Both the person accused of the crime and the accuser would give speeches based on their sides of the story. The individual with the best argument and delivery would determine the outcome of the case. This origin is the source of the two modern usages of the word forensic as a form of legal evidence and as a category of public presentation. In modern use, the term "forensics" in the place of "forensic science" can be considered correct as the term "forensic" is effectively a synonym for "legal" or "related to courts". However the term is now so closely associated with the scientific field that many dictionaries include the meaning that equates the word "forensics" with "forensic science". In forensic biology the most important biological fluids to identify in the form of stains in criminal cases are blood and semen. The other body fluids are saliva, vomit, sweat, urine, faecal matter, human tissues, flesh etc. The evidence of these biological fluids is extremely important in establishing the possibility of guilt or innocence of a person held in connection with a crime. Some other biological examination that are conducted in additional to the above are hair, fibres skeleton remain, dental evidence, poisonous plant etc. In forensic lab we work in three sections:(1) (2) (3) Biology Section Serology Section Toxicology Section

The Policy of Examination The policy behind, solving all forensic science cases is a relatively simple onein theory at least. It was first pronounced at the turn of the century by professor Edmund Locard of Lyons University in the phrase every contract leaves a trace, implying that a criminal always leaves something at the scene of his crime and conversely, takes something away with him. A murderer my leave e body and take away a splash of victims blood, a rapist may unwillingly carry of a pubic hair on his clothing while leaving semen stains behind. All these thingsstains fingerprints, weapons and bullets, scraps of clothing and fibre are grist to miles of forensic science. FORENSIC BIOLOGY: The biology division deals with physical evidence pertaining to living beings, like human and animals, as well as materials of plant origin. This division generally handles the following items of work:I. Bacteriological and entomological examination of exhibits. II. Anatomical examination of human and animal bones, skeletal remains, teeth etc. III. Morphological examination of materials like hair, wool, fiber, wood and wood fragments, seeds, leaf fragments, pollens, micro flora, diatoms etc. with a view to identify the materials. FORENSIC SEROLOGY: The serology division undertakes the following examination:I. Examination of articles stained with blood, semen, sweat, saliva to determine their nature, origin, grouping, DNA profiling etc. II. Determination of paternity through blood groups.

III. Individualisation of blood and bloodstains based on enzymatic studies using latest techniques. IV. Determination origin and grouping of fragments of muscle, skin, bones etc., on objects like bullet, fingernails, etc. FORENSIC TOXICOLOGY: The division deals with:I. The examination of viscera, stomach wash, vomit, etc., to determine poisons of vegetable origin, inorganic salts and metals, synthetic drugs, pesticides, alcohol and other general poisons. II. Examination of powders, pills, capsules, syringes, vials etc. III. Determination of alcohol in blood and urine in drunken driving cases.

FORENSIC SCIENCE SOME BIOLOGICAL EVIDENCES Biological Exhibit (1) Solid Form :- Bone, Hair (2) Liquid Form :- Blood, semen or saliva (3) Bio specimen :- Wood, Pollen grain, diatom (Stoke water) Biological clue material. Ambition is to establish the accused/suspect to the crime or sense of crime. E.g. Blood, Semen, Hair, Saliva, Diatoms etc.

HAIR / FIBRE EXAMINATION

Sampling: 1. The exhibit was spread on a clear white surface under proper illumination. 2. With hand magnifier, loose hair/fiber like sample was carefully located and collected. 3. Hair or fiber found adhering to the exhibit was picked up using a forceps. If the exhibit happened to be a container or an object with crevices, vacuum sweeper with appropriate filter would have been used to collect the hair/fiber. 4. Samples collected in the above manner were individually packed in cellophane/ paper folders and labeled and proper noting was made on the worksheet for their exact location on the exhibit. 5. Each sample was preliminary examined under microscope to note odour, texture and determine whether it is a hair, fiber or indistinguishable at that magnification. 6. Care was taken to note the presence of root bulb or sheath of cells in the hair samples. They were properly preserved for determination of sex or serelogical/DNA examination. Hair Examination: Examination of hair can help in the determination of species of origin, sex, site (part of the body), genetic markers and source by comparison. Different morphological and historical characteristics of hair can be examined under microscope/ stereomicroscope by temporary or permanent mount scale casting, cross-sectioning and micrometric analysis. Temporary Mount: a. Temporary mount of the hair sample was made on a clean slide with distilled water or glycerin. b. Then it was covered with cover-slip. c. It was examined under microscope from one end of hair to the other end for general appearance, length, colour, treatment dye or bleached, presence or absence of root, tip and shaft.Permanent Mount:
5

If the sample is a hair, it may also be cleaned in Xylene and mounted on a microscopic slide as follows: a. Hair was placed on slide in a drop of xylene and permanent mounting medium was added to it. b. A cover-slip was placed on the hair, allowing the medium to spread under cover-slip encasing hair. c. The slide was labeled appropriately and allowed to dry for 48 hours. Following permanent mounting of the hair, it can be examined for different morphological charecteristics and micrometry.

Structure of Hair Cross-sectioning: a. Hair was cleaned in ether:ethanol (1:1) mixture. b. Sample was bundled and dipped in a block of molten wax(at 52C melting point) and was allowed to cool. c. It was then kept in refrigerator for 2-3 hours. d. Cross-section could be taken either with sharp blade or microtone to a thickness of 5-10 micron. e. The section was placed on a thin slide and wax was dissolved with a drop of Xylene on it. f. Permanent mount of the section was prepared and it was then examined under the microscope.

Micrometry: With the help of micrometer measure following distances and calculate different indices: Maximum diameter of the shaft. Scale Count: Number of scales per unit length (100u) Scale Count Index: Diameter of hair in microns/scale count Medullary Index: Maximum diameter of medulla/ Maximum diameter of hair shaft e. Hair Index: Minimum diameter of shaft/ Maximum diameter of the shaft X 100 STANDARDS AND CONTROLS: Animal Hairs: Sufficient samples of hairs from different species should be maintained in a laboratory for reference and comparison purposes. Human Hairs: Hairs of unknown origin may be compared with hairs of known origin to determine the possibility of a common source. For comparison purposes, adequate number of standards (at least 10 strands collected at random) should be examined. General Differences Between Human Hair and Animal Hair Feature Colour Human Hair Animal Hair Often showing profound colour changes and banding Usually less than width of medulla Central or denser towards medulla a. b. c. d.

Relatively consistent along shaft Cortex Occupying most of the width of shaft greater than medulla Distributio Even, slightly more n of towards cuticle pigment Medulla Less than one-third width of shaft. Amorphous, mostly not continuous when present Imbricate, similar along shaft from root to tip

Scales

Greater than one-third width of shaft. Continuous, often varying in appearance along shaft, defined structure Often showing variation in structure along shaft from root to tip

Calculation: M.I of human = width of medulla/width of shaft < 0.3 M.I of non human = width of medulla/width of shaft> 0.3 Sample A: M.I = width of medulla/width of shaft = 6/12 = =0.5 Sample B: M.I = width of medulla/width of shaft = Result: Species of origin Sample A: On the basis of morphological examination, the M.I of sample A was calculated to be 0.5. Hence, it was animal/non-human hair. Sample B: On the basis of morphological examination the M.I of sample B was calculated to be ... Thus, sample B was the hair of any human being.

EXAMINATION OF SALIVA AND SALIVA STAINS The saliva identification in Forensic Investigation is usually done on cigarettes or bidi ends, apparels and in sexual offenses to determine the secretor status. Starch- Iodine Test: Reagent Preparation: 1. 0.5% soluble starch solution (50 mg soluble starch / 10 ml H2O). 2. Lugols iodine solution. a. Iodine 1g b. Potassium iodide 2g c. Distilled Water 200 ml Potassium iodide was dissolved in the water and then iodine was added to it. Procedure: 1. Three tubes were placed in a rack and following were added to it: a) In the first tube, a 5 mm x 5 mm piece of sample to be tested was placed. b) In the second tube, a similar sized unstained control piece was placed. c) In the third tube, 5 mm x 5 mm piece of a known saliva stain was placed. 2. Three drops of soluble starch solution was added to each tube. 3. The tubes were mixed, corked and incubated for 1 hour at 37oC. 4. 2 drops of Lugols iodine was added and colour formed was noted. Observation: A dark blue starch-iodine complex was observed in the second and fourth tubes. The absence of the dark blue colour indicates that the starch has been hydrolyzed and is no longer available for complexing. Therefore, the lack of blue colour is a positive result for amylase activity, indicative of the presence of saliva. [This is not a confirmatory test for saliva].

Precautions: To dissolve the starch, the solution was heated to boil and then the solution was allowed to cool at room temperature. a. Always a soluble starch was used. Hydrolyzed starch was never used. b. Starch solution was prepared fresh daily. A starch solution older than 24 hours was never used.

10

EXAMINATION OF SEMEN & SEMINAL STAINS Physical Examination: Colour: Thick, yellowish white, glairy, opalescent, secretion having a characteristic odor known as seminal. Texture: On touch, seminal stains are starchy. Appearance: Garments sent for forensic examination are usually dirty having variety of stains, in natural light some stains are reddish coloured, while others are brown, yellow or faint grey in colour. These are often mixed with stains of blood vaginal discharge, urine and semen, so as to restrict the investigation to seminal stains only, preliminary examination is done under filtered UV light. The fluorescence of the seminal stains is of a bluish white colour and such stains should be selected for further examination. Confirmatory Test: Microscopic Examination Upon obtaining a positive preliminary test for acid phosphates, the suspected stain can be extracted as follows:

1. Cut a small portion (1 cm2 maximum) of the stain and place in a test tube. 2. Add a few drops of acidulated water to cover the stain. 3. Agitate the stain on a vortex, or in an ultrasonic cleaner bath or manually, by flicking the tube. This will aid in freeing the spermatozoa from the dried stain. 4. When the solution is cloudy, withdraw the liquid with a pipette and place into a disposable 400ul plastic centrifuge tube and centrifuge in a microfuge for 30 seconds. 5. Carefully withdraw the supernatant, which contains soluble group-specific substances, enzymes and other solutes from seminal plasma. 6. Collect the button of cellular and other insoluble components from the tube and place on a clean-labeled microscope slide. 7. Fix in dilute H2SO4 acid and dry. It is now ready for staining.
11

Gram Staining Reagent Preparations: 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. Mix Solution A and Solution B to form Reagent # 1 Reagent #2: Grams iodine Iodine Potassium Iodine Distilled water

1g 2g 300 ml

Reagent # 3: Decolorizer 95% ethanol. Reagent # 4: Safranin solution. Safranin (2.5% in 95% ethanol) Distilled water

10 ml 300 ml

Procedure: 1. The smear or stain extracted was fixed to the slide by gentle heating or chemical fixative. 2. Crystal violet Solution (Reagent # 1) was added to slide for 1minute. 3. It was then rinsed with tap water (some of the material on the slide remained as violet colour). 4. Grams iodine Solution (Reagent # 2) was added to the slide for 1minute. This fixed the crystal violet to the tissue). Rinse with tap water. 5. Decolorizer (Reagent # 3) was added and the slide was tilted back and forth, watching most of the violet colour wash away. This took about 10 seconds. 6. It was then rinsed with tap water and Safranin Solution (Reagent # 4) was added to it and was allowed to remain for 1minute. 7. Lastly, it was allowed to air dry and was examined with oil immersion at 1000X.

12

Observation: Gram positive protein will stain violet, gram-negative protein will stain red. Spermatozoa will appear as differentially stained purple bodies, somewhat oval in shape with a clearly discernible acrosomal cap.

13

EXAMINATION OF BLOOD & BLOODSTAINS Blood: Blood is a extracellular fluid which is made of connective tissues. Blood is made up of cells (red blood cells and white blood cells) plus platelets in a yellowish liquid called plasma. Analyzing Genetic Variation in Blood Forensically: a. Hemoglobin (RBC): Peroxidase-like activity can cleave Hydrogen Peroxide. b. Blood Group Antigen (RBC): ABO groups (on surface of RBCs) c. DNA (WBC): Found in nucleus of WBCs d. Proteins (Plasma): Serum used in species testing

Physical Examination: In natural light examination of exhibits for brown, reddish brown stains, powder or crystals of reddish brown colour, these areas should be demarcated. In case of absence of clear and visible stains, washed stains should be examined under 230-269 nm frequency UV light. Presumptive Test: These suspected bloodstains; contaminated materials should be tested for positive for blood.

SCREENING TEST FOR BLOOD: For screening test of blood, we use Tetra Methyl Benzidine (TMB) test. Reagent Preparation: Acetate Buffer is formed of : a. Sodium Acetate - 5.0 gm b. Glacial Acetic Acid 43.0 ml
14

c. Deionized water 50.0 ml

Working Solution: a. TMB 0.4 g b. Acetate Buffer 20.0 ml

These solutions were mixed, filtered and stored in brown coloured bottle in refrigerator.

Procedure: 1. A cutting or swabbing of the stain was placed on filter paper or spot test paper. 2. A drop of TMB solution was placed on the stain and followed by a drop of 3% hydrogen peroxide. Observation: An intermediate blue green colour gave a positive test for hydrogen peroxide activity, indicative of hemoglobin. Standards and Controls: A known bloodstain and unstained control must be tested.

CONFIRMATORY TEST FOR BLOOD: Takayama Test: For confirmatory test for blood, we use the Takayama Test. A. Reagent Preparation for Takayama Test: a. Standard glucose solution(100 g glucose/ 100ml water) 3 ml

15

b. 10% NaOH 3 ml c. Pyridine 3 ml d. Distilled water 7 ml B... Procedure: a. The material to be tested on microscopic slide was placed and covered with a cover slip. b. A drop of Takayama reagent was added and allowed to flow under the cover slip. c. Slide was warmed gently on a hot plate at 65C for 10-20 seconds. d. It was then allowed to cool and then was observed under microscope at 100X.

B. Observation: Appearance of pink needle shaped crystal of pyridine was the positive reaction for hemoglobin.

Teichmanns Test: A. Reagent Preparation: KCL + KBr KI 0.1g each 0.1g 100 ml

Glacial Acetic Acid

It was mixed and stored in stoppened bottle.

16

B. Procedure: a. The material to be tested was placed on a microscopic slide and covered with a cover slip. b. The reagent was allowed to flow under the cover slip. c. The slide was warmed gently on a hot plate at 65C for 1020 seconds. d. It was allowed to cool and then was observed under microscope.

C. Observation And Result: Appearance of brown rombohedron shaped crystal of Ferroproto prophyrin chloride showed the positive reaction for hemoglobin.

17

SEROLOGY
Serology allows the forensic scientists to segregate these bodily fluids when found at the scene of the crime and then perform a variety of tests on them in order to identify where these fluids originated from - or most importantly - who they came from. Determination of the type and characteristics of blood, blood testing, bloodstain examination, and preparation of testimony or presentations at trial are the main job functions of a forensic serologist, who also analyzes semen, saliva, other body fluids and may or may not be involved with DNA typing. Serology, in addition to examining and identifying blood, is used to identify and categorise semen, saliva, sweat and even human faeces. This can be achieved in the instance of faeces as it is covered in a mucus membrane to enable expulsion from the body. Serology also has a use in proving if unlawful sexual intercourse has taken place; this has become a necessary element of forensic science given the rise in sexual assaults and cases of rape. The processes used by a Serologist can help time intercourse and also help prove that unlawful intercourse actually took place. Secretors and Non-Secretors It is important to note that although a large percentage of the population are classed as 'secretors' there are a smaller percentage of people who are nonsecretors. Secretors exhibit elements of their blood's protein when they secrete other bodily fluids whilst non-secretors will not have levels of protein from their blood in their bodily fluids. Testing the bodily fluids of secretors will reveal a result but non-secretors make it difficult for Serologists to gain any results so blood from these individuals must be tested in order to provide any level of positive result. Again it is important to note that these procedures are used when other means of identification yield no results and although these tests may prove accurate other means of identification should be used, leaving this kind of scientific evidence to provide additional weight to any legal proceedings. Serology concern with detection, origin and grouping of species.

18

DETERMINATION OF ORIGIN OF GIVEN BLOOD STAIN Once a biological evidence has been identified , it is necessary to determine whether or not it is of human origin; and if of non human origin , then to what species it belongs. The species specific proteins in the blood stains or other body tissues may be identified with the help of species specific antibodies. In this antigen and antibody of same species of origin diffuse towards each other and precipitin arc forms.For determination of the species of origin, we perform Precipitin tube method, Double diffusion and Cross gel electrophoresis.

DOUBLE DIFFUSION METHOD In Double diffusion method, both of the reactants, antigen and antibody diffuse towards each other in the gel; and when an antigen combines with its specific antibody at optimum proportion , a precipitin arc forms. A sample extract of interest (for example human cells harvested from tonsil tissue) is placed in one well, and sera or purified antibodies are placed in another well and the plate left for 48 hours to develop. During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells. Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form what is known as an immune complex. This immune complex precipitates in the gel to give a thin white line, which is a visual signature of antigen recognition. The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple wells with different antibodies or mixtures of antibodies, and antigen-antibody reactivity can be seen by observing between which wells the precipitate is observed. When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected. The zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely). Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of IgM there

19

is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed. PROCEDURE :1. Weigh 1g of agar and put in a beaker containing 100 ml of normal saline. 2. Heat the solution while shaking. 3. When the agar gets dissolved completely, pour it over in a properly levelled disposable glass slide to make a 1-2 mm thick agar layer. Wait until the agar solidifies. 4. Punch wells in gel, each about 5 mm apart. 5. Seal the bottom of punched wells with dilute agar. 6. Fill the central well with tissue extract and glass peripheral wells with different antiserum for species of origin or vice versa (Antihuman, Anticow, Antigoat etc.). 7. Cover the glass slide and keep in a moist chamber for overnight. 8. Examine gel for the presence of precipitin arcs. PRECIPITIN TUBE METHOD A serological test using a precipitin reaction to detect the presence of a specific antigen; specifically : a test used in criminology for determining the human or other source of a blood stain The precipitin reaction is based upon the interaction of antigen with antibody leading to the production of antigen:antibody complexes. In the precipitin reaction, varying amounts of soluble antigen are added to a fixed amount of serum containing antibody. As the amount of antigen added in

In the zone of antibody excess, each molecule of antigen is bound extensively by antibody and crosslinked to other molecules of antigen. The average size of antibody-antigen complex is small; cross-linking between antigen molecules by antibody is rare. In the zone of equivalence, the formation of precipitin complexes is optimal. Extensive lattices of antigen and antibody are formed by crosslinking. At high concentrations of antigen, the average size of antibody-antigen complexes is once again small because few antibody molecules are available to cross-link antigen molecules together.

20

PROCEDURE :1. Take six precipitin tubes and place them vertically in a precipitin tube stand and label. 2. Put a drop of a blood stain extract in the tubes. 3. Carefully add 1 drop of antiserum for species origin along the walls of tube and leave undisturbed for half an hour in room temperature. 4. Examine for a white ring at the interface of two solution.\ DETERMINATION OF ABO BLOOD GROUPING Blood group of every person fall into four blood group A,B,AB and O identified in 1900 by Landsteiner, The ABO blood grouping system is a function of the red blood cells and the presence in them of a substance known as agglutinogen. The RBCs of each individual are studded with antigen give rise to the specific blood group in the individual. The presence of these specific antigens can be detected by specific antibodies. The antibodies are capable of precipitating these specific antigens thus resulting in yhe agglutination of RBCs. The antiserum with which the RBCs of the blood shows agglutination the blood has that particular group. In the ABO system the dried stain survive for many years and retain the capability of combining with specific antibodies.Formation of antigens-antibody complexes is the basis of all methods employed in the detection of red cells antigens in blood stains. GROUPING METHOD OF BLOOD STAIN BY ABSORPTION ELUTION

PROCEDURE :1. Take three clean and dry test tubes and mark them A, B and H. 2. Cut the blood stain and put about 2 sq mm or 2-5 mm long thread in each test tube. 3. Dip the fabrics in anti-A serum, anti B serum and anti- H lectin respectively and keep at 4C for overnight. 4. Then remove the antiserum and give 3-4 washing with chilled normal saline. 5. After the last wash remove whole of the normal saline. 6. Add 1 drop of 2% A,B and O indicator cells in the respective tubes.
21

7. Plug the test tubes with cotton and keep into the incubator at 56C for 15 mins. 8. Shake it for 10 mins in the shaker.Examine the clumping macroscopically and macroscopically.

AGGLUTINATION IN CAVITY A + + B + + + H

BLOOD GROUP

A B AB O

PRECAUTION :1. Washing should be done with ice chilled normal saline. 2. At the time of elution , only 1 drop of normal saline is required.

22

DETERMINATION OF BLOOD GROUP FROM SALIVA/SEMEN BY ABSORPTION INHIBITION METHOD PROCEDURE :1. Prepare the dilution of antiserum of its penultimate titre (if the titer is 32 then prepare a 16 dilution by taking 1 drop of the serum and adding 15 drops of normal saline to it). 2. Take a clean and dry cavity tile and mark three cavities A,B and H and place 1 drop of suitable dilution of anti Aserum, anti B serum and anti H lectin to the respective cavities. 3. Add 1 drop of the extract in each cavity.Shake and keep at 4C for 2 hrs. 4. Add 1 drop of 2% indicator cells in the respective cavities and keep at 4C for half an hr. 5. Shake the tile and examine the content for the presence of agglutination both macroscopically and microscopically.

AGGLUTINATION IN CAVITY A + + + B + + + H + -

SECRETOR STATUS NON SECRETOR SECRETOR SECRETOR SECRETOR SECRETOR

BLOOD GROUP

A B O AB

23

TOXICOLOGY Toxicology: It is a Greek word derived from two words toxicon and logos. The word toxicon means aero poison and logos means science and study respectively. Toxicology is that branch of science which deals with property, action, toxicity, fetal dose, detection, estimation and treatment of poison. Toxicology is mainly divided into three branches: a) Environmental toxicology: It includes the study of those chemicals and gases which produce harmful effect on human and animal body if it comes in their contact directly or indirectly. Ex: Harmful effect produced by water pollution, air pollution, radioactive decay sewage, etc. b) Economic toxicology: It includes the study of those chemicals which are used by human beings for specific object so that better economic condition or profit could be obtained by that object. Ex: Toxic effects due to food preservatives, edible dyes, fertilization and use of insecticides. c) Clinical toxicology: It deals with human disease caused by air due to abnormal expose to chemical substance (drugs). Ex: Overdose of any drug or continuous use of any drug produce harmful effect to the body. Forensic Toxicology: It is closely related with clinical toxicology. It deals with medical and legal aspects of harmful effects of chemicals on human beings. i. ii. Medical aspects: It deals with diagnosis and treatment of developed harmful effects. Legal aspects: It includes all those information which are related with contact and entry of poison with human being.

POISON: Poison is a Latin word derived from potus which mean a drink material which is used to produce harmful effect on human beings or animals and produce death. It is a substance (solid, liquid or gas) which if introduced
24

into living body or brought into contact with any part will progigitable duce ill health or death by its constitutional or local effects or both. According to Paracelsus, no substance is poisonous, but its excess amount makes it poisonous. It means these substances in excess amount are poison.

CLASSIFICATION OF POISON: According to toxicology analysis, poisons are classified into several classes: i. Volatile Poison: Ex- Ethyl alcohol, Acetone, Chloroform, etc. ii. Non-volatile Poison: It is the main classes of poison which is sub-divided into :a. Acidic Extract: Ex- Pheno. b. Basic Extract: All drugs which is basic is poisonous. iii. Metallic Poison: On this category, all metals are present for Ex- Zine, Sodium, Potassium, etc. iv. Anions(Anionic Poison): Chloride, Bromide, Synides, Phosphate, Sulphate, & all anions. v. Pesticidal Poison: a. b. c. d. e. Arsono phaspho: Ex- M. Arsono chloro: Ex- BHC, DDT. Carbamet: Ex-Begon. Pyrithraid: Ex- Pyrithim plants. Vegetable poison: Ex- Dhathura, Bhang, etc.

On the basis of chief symptoms produced on consumption of poison, poisons are classified into following three categories:
25

i. Corrosive Poison ii. Irritant Poison iii. Systemic Poison Corrosive poison: these poisons cause decomposition or deformation of skin or tissue or contacted area. ExStrong acids HCl, H2SO4, HNO3, HCN, CH3COOH, etc. Strong bases- K2CO3, Na2CO3, KOH, NaOH, etc. Irritant poison: These poisons cause pain, irritation, inflammation on the affected area. Ex- Inorganic irritant Cl-, Br-, I-, P-,(non- metallic) and As, Sb, Bi, Pb, Zn, Hg (metallic). Physical irritant hair, glass, diamond dust, dry sponge, etc. Organic irritant castor, croten, abrus, calotropus (vegetable), snakes, insects, cantharida, venoms (animals), etc. Systemic poison: These poisons affect the nervous system. They after absorption exert their poisonous effect even when well diluted in water. FLAME TEST FOR DIFFERENT METALS: Flame test is a method used to find out different metals by heating the metals directly in the flame and change in colour produced in the flame can be used to identify the metal. Following are the metals which can be identified by different colours produced in the flame: a. Potassium: when Potassium is heated directly in flame, then crimson red colour flame is produced. b. Sodium: When sodium chloride is heated in flame then the colour of the flame becomes golden red due to presence of sodium in it. c. Calcium: When any calcium salt or compound is heated in flame then the colour of the flame becomes brick red due to presence of calcium in it.

26

NARCOTICS DRUGS AND PSYCHOTROPIC SUBSTANCES Narcotics Drugs and Psychotropic Substances (NDPS) is that part of forensic chemistry which finds out the drugs and psychotropic substances which according to the NDPS Act are not allowed to be sold purchased or consumed. For example: Ganja, Charas, Heroin, Poppy plants, Thebaine, Papaverine, etc. Cannabis sativa: Cannabis sativa is a plant widely distributed throughout the temperate and tropical zones of the world and most countries have reported illegal growth and traffic of the herbal products. The fruiting and flowering tops and leaves of the cannabis plant contain significant quantities of the psychoactive constituents (e.g. tetra hydro cannabinol); they are known as the drug containing parts. These parts may be stripped from the plant while it remains growing and the central stem and main side stems of the plant are not removed and play no part in the production of illicit cannabis productsWhole plants can be dried while suspended upside down and when dried, the drug containing parts of the plant are stripped from the central and main side stem. A wide range of herbal presentations can also be made depending on the process subsequently used on the dried material. Ganja: Ganja is a flowering and fruiting top of the female Cannabis Sativa plant. It is a hemp plant. Ganjais also known as Baba Sulpha Dope, etc. Charas : Charas is resinous exclude of flowery and fruity top of female Cannabis Sativa plant. Charas is also known as HASHIS. Bhang: In the absence of flowering and fruiting part of plant Cannabis Sativa, bhang is considered as a greenish brown leaves along with stems. It is found in both male and female plant. It is not punishable under NDPS Act. The chief intoxicating ingredient in Cannabis plant is Tetra Hydro Cannabinol(THC.) Heroin: Heroin is basically a diacetyl derivative of Morphine. It is a chemical derivative of poppy plants. A milky substance is extracted from poppy fruit and it is reacted with acetic anhydride to form heroin. Thus. It is a drug punishable under NDPS Act. Heroin is also known as Brown Sugar Smack, etc.

27

Opium: the main alkaloids of opium which is detected in the chemical test analysis i.e. a. b. c. d. e. Morphine Codine Narcotine Thebaine Papavarine

TESTS FOR HEROIN: Screening Test (Colour test): I. Marquis Test: Procedure: a. Firstly, sulphuric acid and formaldehyde was mixed in a beaker in 9:1 ratio. b. Then the sample was put in the vertical tiles and the above solution made was added on it. Observation: Firstly, purple colour was developed, then it was converted into brown colour and finally it became blue due to the solution used. Result: The change in the colour of the sample from purple to blue indicated the presence of heroin in the sample. II. Frohds Test: Procedure: a. A solution of 1% Ammonium Molybdate salt and 99% Sulphuric acid was made. b. Sample was dissolved in this solution. Observation: Firstly, greencolour was developed which then converted to voilet and finally to pink.
28

Result: This change of colour confirmed the presence of heroin in the sample. Confirmatory Test for Heroin: Thin Layer Cromatography or TLC test is the confirmatory test for Heroin. 1. Preparation of TLC plate: TLC plate is made up of silica gel G and water in 40:20 ratios. It is then kept inside oven for 20-30 minutes so that it can get activated and can be used. 2. Preparation of System Chamber: Firstly, chamber was washed with chloroform. Then 90 ml chloroform and 10 ml methanol was put inside it (instead of 90ml chloroform and 10 ml methanol, 90 ml ammonia and 10 ml methanol can also be used ). Then it was shaked well for proper mixing and was put as it is for 30 minutes to 1 hour. 3. Spraying Reagent: Spraying Reagent used to test Heroin is Dragondroff. The solutions used to make this reagent are as follows: Dragondroff spraying reagent was made by mixing the following: a. Bismuth sub-nitrate solution 10 m Bismuth sub-nitrate solution : 2 gram of Bismuth sub-nitrate solution was dissolved in 100 ml distilled water and 25 ml glacial acetic acid was added to it. 10 ml of this solution was taken to make the reagent. b. Potassium Iodide solution 10 ml Potassium Iodide solution: 40 gram potassium iodide was added to 100 ml distilled water.10 ml of this solution was taken for use. c. Distilled water d. Glacial Acetic Acid 60 ml 20 ml

29

4. Procedure:a. Samples were extracted with methanol in a test-tube so that sample could get dissolved into it. b. Then spotting was done by placing the samples in the TLC plate. c. Then this plate was put inside the system chamber for 45 minutes to one hour for running the sample. d. After that plate was taken out and spraying was done over it by using developing reagent or spraying reagent Dragondorff reagent. 5. Observation: Two spots of orange colour developed on the TLC plate. 6. Result: Orange colour spots which developed on the plate confirmed the presence of heroin in the sample.

Orangish brown spots of herioin.

30

THIN LAYER CHROMATOGRAPHY(TLC): Ganja, Charas and Bhang can be detected by Thin Layer Chromatography or TLC. 1. Preparation of TLC plate: TLC plate is made up of silica gel G and water in 40:20 ratios. It is then kept inside oven for 20-30 minutes so that it can get activated and can be used. 2. Preparation of System Chamber: This chamber is prepared by putting hexane and acetone in the ratio 80:20 respectively inside it and then it is put as it is for 45 minutes to one hour. 3. Preparation of Spraying Reagent: Spraying Reagent used to test bhang, ganja and charas is Fast Blue B. It is prepared by dissolving a very small amount of Fast Blue B in 100 ml distilled water and 2 to 3 drops of methanol is added to it. It is shaked well for proper mixing. 4. Procedure:a. Samples were taken and dipped into hexane in a test-tube so that sample could get dissolved into it. b. Then spotting was done by placing the samples in the TLC plate. c. Then this plate was put inside the system chamber for running the sample for 45 minutes to one hour. d. After that plate was taken out and spraying was done over it by using developing reagent or spraying reagent. 5. Observation: Purple colour was developed on TLC plate with different spots which showed the presence of bhang, ganja and charas in the sample due to Tetra Hydro Cannabinol (THC) in it.

31

Purple colour spots of tetra hydro cannabinol was detected .

32

CONCLUSIONS: In forensic science laboratory we examine the suspect of crime by investigation of biological (semen, saliva, and blood), toxicological (different poison/ toxin) and narcotic (ganja, charus, bhang, heroine etc) exhibit by different method and confirm the actual victim of crime. This can be done by examination of hair, blood grouping, bone examination, detection of nature of poison and by examination of narcotic sample.

33