Forensic Biology - Biological evidences

The slides explain in detail about Forensic Biology and how it works.

1. Forensic Biology Unit 1

2. Nature of biological evidence Biological evidence is commonly seen in criminal cases. Human beings are biological beings and therefore possess and secrete biological tissues / fluids. Blood , semen, saliva, tears, urine, feces, menstrual blood, sweat are common examples of biological products which can be encountered in crime cases as evidence. They help prove the corpus deliciti (that a crime happened) and also proves the chain of events that led to the crime. Before learning the collection and analysis of these biological evidences, it is important to understand their role in the human body, their functions and significance as evidence .

3. Human Body – Anatomy vs. Physiology Simply put, anatomy is the study of the structure and identity of body parts, while physiology is the study of how these parts function and relate to one another. Anatomically bones are arranged in a particular manner in the human body. Physiologically they provide support and rigidity to the body. We will start off by understanding a little anatomy & physiology of each human body system to understand the source of biological evidence and their biochemistry.

4. Circulatory system / Cardio-vascular system

5. Lymphatic system

6. Digestive system

7. Renal system

8. Nervous system

9. Skeletal system

10. Epidermal system

11. Blood Blood is a connective tissue in the body and helps to transport oxygen, nutrients, enzymes, hormones and waste products in the body. It is more viscous than water but it is mostly constituted with water. The proteins and cells add to its viscosity. Blood is fractioned into cells (45%) and plasma (55%). Plasma being the liquid part of blood carries the proteins, enzymes and hormones etc. in it. Plasma is a straw colored liquid in which the cells are carried along. Anticoagulated blood in the body is fractioned as cells and plasma – the clotting factors are still active in plasma. When the blood has clotted – the clotting factors are missing, blood separates as cells and serum. The cellular fraction of blood is composed of 3 different cell types – the Red Blood Cells (RBCs / erythrocytes), the White Blood Cells (WBCs / leukocytes) and the platelets (thrombocytes). The RBCs carry hemoglobin and help in oxygen carrying. The WBCs help in cell mediated immunity. The platelets help in primary clot formation during minor injuries.

12. Hemoglobin as target molecule Plasma Vs serum Vs antiserum - YouTube

13. Hemopoiesis Most of the hemopoiesis happens in the spleen, bone marrows and thymus. Destruction of blood cells happens in spleen and liver. The inconsistency in these functions is what causes anemia, leukemia and other blood related disorders.

14. Erythrocytes RBC’s) RBCs are produced in the bone marrow and spleen. They are anucleated – don’t contain a nucleus, and are biconcave shaped (doughnut shaped without the hole) – this shape helps in larger surface area for oxygen carrying and also to flow easily in thin blood vessels. RBCs give blood the red color. Hemoglobin is present in many other animals also and RBCs are only a marker for blood – not particularly for ‘human’ blood. The average lifespan of RBCs is 120 days and it keeps getting regenerated in the marrow and spleen.

15. Erythrocytes RBC’s)

16. Hemoglobin Hemoglobin is a globular protein – shaped like a globe, and is formed from four sub-unit proteins, each with a ‘ haem ’ group (Fe) in the middle. The haem group is able to reversibly bond to oxygen which is why hemoglobin is so helpful for transporting oxygen around the body. Hemoglobin is what gives the RBCs the red color. Hemoglobin levels are relative to iron levels and protein levels in the body. Lack of hemoglobin is termed as ‘anemia’.

17. Hemoglobin

18. Leukocytes WBCs are produced in the bone marrow or lymph nodes. They are termed ‘white due to their absence of pigmentation. They are bigger cells than the RBCs and are nucleated. Few WBCs contain granules in their cell cytoplasm and few don’t. So they are classified as granulocytes and agranulocytes . Granulocytes can be Neutrophils (50-70% - Bluish green granules), Eosinophils (1-3% -Orange red granules) and Basophils (0-1% - Purple granules). Agranulocytes include Lymphocytes (20-40% - round nucleus with scanty cytoplasm) and Monocytes (Horse shoe shaped nucleus with abundant cytoplasm ). Their major function is to provide cell-mediated immunity in the body. Forensically they are significant because of the presence of nucleus. This is the only blood cell type which contains nucleus, and therefore nuclear material viz. DNA. WBCs are the source of DNA in blood evidence.

19. Leukocytes

20. Thrombocytes Platelets are small fragments of cellular material floating in the plasma of blood and gets transported all across the body. They are anucleated fragments. They are much smaller than RBCs. Their major function is to form a platelet plug to initiate clotting when there is an injury in the blood vessel. This keeps us hemodynamically stable and stops blood loss. Without sufficient platelets blood clotting would be slow and could be fatal.

21. Thrombocytes

22. Tests for blood Tests for blood target the hemoglobin component in blood. WBC’s serve as DNA source. Platelets have no forensic significance.

23. Blood vessels What are Blood vessels? | Blood Circulation in Human being | Biology | Letstute - YouTube

24. Hemoglobin as target molecule Human blood Animal Blood Color Red Red Hemoglobin Present Mostly present except in fish, insects etc. WBCs Present Less predominantly present Blood group antigens Present; Mostly ABO antigens Present, but the types of antigens are more in number DNA Present in WBCs Present in RBCS & WBCs

25. Hemoglobin as target molecule Hemoglobin is a globular protein with a ‘ haem ’ group (Fe) in the middle. Fe molecule is target for medical hemoglobin estimation. In Forensic Science, we test for the Fe molecule along with the effect of the globin protein in it – to be little more specific. The tests that we use are Benzidine test Phenolphthalein test Takayama test Teichman Test Spectrocopy test Blood smear study Blood Pattern Analysis DNA

26. Benzidine test Benzidine Test - YouTube

27. Phenolphthalein test

28. Takayama test

29. Teichman test

30. Spectroscopy test

31. Leishman stain Leishman stain is a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure. Leishman stain is a differential stain that is used to variably stain the various components of the cells

32. Leishman stain Principle of Leishman's staining(Clear overview about principle). - YouTube

33. Blood Pattern Analysis BLOOD PATTERN ANALYSIS is the examination of the shapes, location and distribution patterns of bloodstains, in order to provide an interpretation of the physical events which gave rise to their origin. Gravity, Potential & kinetic energy and geometrical principles are used to study the patterns.

34. Factors that can b e ascertained by Blood Pattern Analysis Height Direction / Angle Pressure Surface Source Volume Force

35. Height - BPA

36. Direction - BPA

37. Direction - BPA

38. Angle of fall - BPA

39. Angle of fall - BPA Sin (Angle) = Width (a) / Length (c) In this case Sin (Angle) = 1.5/3.0 = 0.5 Therefore Angle = 30°

40. Pressure - BPA

41. Surface - BPA Glass surface Linoleum flooring Wood or concrete Wallpapered wall Tiled wall

42. Source - BPA Arterial Blood / Arterial Spurt Venous Blood

43. Source - BPA Arterial Blood / Arterial Spurt Venous Blood Small drops of blood Forms a wavy pattern Volume of blood is more The colour of blood stain is brighter Bigger drops of blood Does not form a wavy pattern Volume of blood is lesser The colour of blood stain is duller

44. Volume - BPA Less Bleed Pool of blood formation

45. Force - BPA Low Velocity Medium Velocity High Velocity

46. DNA Profiling Nucleus of every living cells contains nuclear material, which are our DNA and derived from our biological parents. DNA profiling is the means of detecting distinctive DNA sequences in human DNA to compare and identify from evidence DNA. Used for identification of individual from blood evidence seen in Scene of Crime, for paternity disputes etc.

47. Review of blood Blood is a connective tissue. Carries oxygen, nutrients and wastes. Cells ( RBC+WBC+Platelets ) and plasma. Hemoglobin is the oxygen carrier. Tests we perform for blood.

48. Semen & its physiology Semen or seminal fluid is the thick fluid that comes from men’s penis when they ejaculate during sexual activity. It carries sperm out of a man’s body so it can fertilize an egg and create an embryo. While sperm is the principle component of semen, it is not necessarily the only component. Sugars, enzymes and proteins are part of seminal fluid.

49. Male reproductive system The external anatomy includes the penis and the testes (covered in the scrotum). The internal morphology includes The testes (testicles) which is made up of seminiferous tubules ( sertoli cells & Leydig cells) and connected to the penis by the vas deferens (Vasectomy is the process of discontinuing the vas deferens and is a means of contraception). The sertoli cells produce the sperms and the hormone testosterone. The seminal vesicle is further internally situated and secretes its contents into the penis and along with the seminal fluid. Fructose and amino acids are secreted by the seminal vesicle. This is the maximum fraction of human semen. The prostate gland, situated below the seminal vesicle produces acid phosphatase enzyme, prostaglandins and Prostate Specific Antigen (PSA). The cowper’s gland / bulbourethral gland is situated below the prostate gland and secretes galactose and mucus. The fractions in semen are – testes (2-5%), seminal vesicle (65-75%), prostate gland (25-30%) and cowper’s gland (0-1%).

50. Male reproductive system

51. Semen On an average men secrete 3-4ml of semen per ejaculation. The principle component – spermatozoa is actually a male reproductive cell (gamete) involved in reproduction. Sperm is a unicellular unit, with a tail - known as flagellum and it helps to be motile. The head of the sperm, known as acrosome contains cellular matrix and a nucleus. This nucleus has 23 chromosomes ( duploid ). Similarly the egg in the woman has 23 chromosomes ( duploid ). During reproduction, the sperm gives its 23 chromosomes (22 autosomes + 1 sex chromosome) and the egg gives its 23 chromosomes (22 autosomes + 1 sex chromosome ). These together make the 46 chromosomes of the zygote (child ). The neck of the spermatozoa contains ATP and energy proteins to keep the sperm functional until fertilization.

52. Semen

53. Semen

54. Semen

55. Semen

56. Constitution of semen Part of the male reproductive system Constituent Seminiferous tubules (Testes) 0-2% Spermatozoa Testosterone Seminal Vessicle 65-75% Fructose Amino acids Prostate gland 25-35% Acid Phosphatase Prostaglandins PSA Cowper’s gland 0-1% Galactose Mucus Glans penis (1-5%) Smegma

57. Specimen sent for semen analysis Clothes of victim Clothes of accused Bedsheets Vaginal swabs Vulval swabs Anal swabs Axillary swabs Nipple / areola swabs Pubic hair Condoms Control semen

58. Specimen sent for semen analysis Collection in all sexual assault cases are done by a professionally trained forensic medicine degree holding doctor in the presence of a official deputed form Women & Children welfare department. The collection is prescribed to be done using a Sexual Assault Evidence Collection Kit (SAECK). SAECK is approved for one time use per kit. It is designed in such a way to ensure DNA extraction as well as biological and serological efficiency.

59. Tests performed for semen Touch examination Dried semen stains have a starchy feeling and are easily identifiable.

60. Tests performed for semen UV light examination Seminal stains fluoresce as bluish-yellow colour under UV light .

61. Tests performed for semen Acid phosphatase test Seminal stains when reacted with Sodium alpha- naphthyl phosphate and Naphthanil diazo blue produces a purple colour

62. Tests performed for semen Florence test Seminal stains with Potassium Iodide and Iodine form dark brown heamin crystals of choline periodide resembling wooden splinters .

63. Tests performed for semen Barberio’s test Seminal stains with saturated picric acid solution forms needle shaped spermine picrate crystals

64. Tests performed for semen Prostate Specific Antigen [PSA] Commercially available kit cards are available to pick up presence of PSA. PSA is present even in vasectomized individuals.

65. Tests performed for semen Microscopy Seminal stains can also be mounted on microscopic slide, stained with Gram’s stain and viewed under microscope for presence of spermatozoa. Human spermatozoa is characterised by a DNA rich head, a mid-piece mitochondria and a long tail.

66. Tests performed for semen Gram staining is done in microbiology to differentiate between gram positive and gram negative bacteria. The spermatozoa under gram stain is easy to pick up as it has a gram positive head & neck and gram negative tail.

67. Tests performed for semen

68. Tests performed for semen Grouping Seminal stains also carry blood group antigens, in case of secretors. The blood group antigens are secreted in all body fluids of secretors . Diluted Antisera is used for this purpose.

69. Saliva Saliva is a colorless, opalescent fluid that is constantly present in the mouth of humans. It is composed of water, mucus, proteins, mineral salts, and amylase. As saliva circulates in the mouth cavity it picks up food debris, bacterial cells and epithelial cells of the mouth lining. One to two liters of saliva is excreted daily into the human mouth. Three major pairs of salivary glands and many smaller glands are scattered in the oral cavity. The major function of saliva in the body is to initiate digestion of carbohydrates. It achieves this by the enzyme ‘salivary amylase’ present in saliva. Other functions include lubrication of the digestive tract and to improve the sense of smell and taste.

70. Saliva

71. Saliva Salivary Amylase – Enzyme Produced by the salivary glands and has the ability to break down longer chain carbohydrates into smaller chain carbohydrates. The major dietary long chain carbohydrate is starch. Salivary amylase breaks down starch to maltose, lactose and glucose.

72. Saliva Salivary amylase is also the target molecule for forensic tests. Starch-Iodine test The suspected saliva stain is placed in a test tube. 3 drops of soluble starch is added to it followed by a drop of Lugol’s Iodine. Starch reacts with the Iodine and forms a starch-iodine complex which has a blue – black colour . The contents are incubated at 37°C for 1 hour and every 10 minutes the tube is observed for colour change. Result: If saliva is present in the suspected stain the salivary amylase enzyme digests the starch and breaks the starch-iodine complex and changes the colour to the hay brown colour of Iodine. (Picture below) If saliva is absent in the suspected stain, the blue-black colour persists.

73. Saliva

74. Saliva Other tests such as gel diffusion using starch gel and iodine counter stain are also used to detect saliva. Saliva stains also carry blood group antigens, in case of secretors. And can be grouped.

75. Saliva Forensic significance of saliva as evidence Sexual assault cases – usually as bite mark Suicidal hanging cases. Cigarette butts, handkerchiefs etc. in stalking and kidnapping cases.

76. Tear Tear is a clear liquid secreted by the lacrimal glands (tear gland) found in the eyes of humans. Tear is a secretion of emotion secreted by humans to express their feelings. It is made up of water, electrolytes, proteins and mucins. The ratio of each component varies at different intervals in the human body.

77. Tear

78. Tear Tests for tears Lactoferrin is the target molecule. Specific testing kits with patented tech are available for testing for Lactoferrin .

79. Urine Urine is a watery, typically yellowish fluid stored in the urinary bladder and discharged through the urethra. It is one of the body's chief means of eliminating excess water and salt, and also nitrogen compounds such as urea and other waste substances removed from the blood by the kidneys. During food metabolism many nitrogenous waste products are formed in the body which when accumulated can cause toxicity and electrolyte imbalance. They need to be removed from the body. The renal system includes the kidneys (2 nos.) and the renal apparatus which filters the waste products out of blood and excretes it as urine.

80. Urine

81. Urine Tests for urine UV examination - Urine stains fluoresce as yellow/pale blue in UV light. Flame test - On heating gently over a flame, the characteristic odour of urine may be detected. Urea Nitrate Crystal test - An aqueous extract of the stain when reacted with one drop of conc. Nitric acid on a slide forms hexagonal urea nitrate crystals.

82. Urine Creatinine test – One drop of picric acid is added to stain followed by 5% Sodium hydroxide – Brown/orange color shows presence of creatinine. Urine stains also carry blood group antigens, in case of secretors. And can be grouped .

83. Feces Feces is the solid or semisolid remains of food that was not digested by the digestive system. It contains metabolic waste, undigested material, enzymes, gut bacteria, epithelial cells etc . It contains undigested food material, digestive enzymes and pigments . Test for feces A small of amount of stain scraping is mounted on a slide with a drop of Lugol’s Iodine and observed microscopically for undigested vegetative matter, muscle fibres , etc. Urobilinogen test: the stain is reacted with mercuric chloride, zinc and amyl alcohol. Fecal stains produce a green fluorescence of zinc- urobilin . Can be grouped.

84. Menstrual blood Menstrual blood is the product of menstruation in menstruating women. On an average 35 ml of menstrual blood is secreted by the female reproductive system during a single menstruation cycle. Menstruation is the regular discharge of blood and mucosal tissue from inner lining of the uterus. It must be noted that a woman starts menstruating around 12-15 years of age (menarche) and stops menstruating around 45-55 years of age (menopause). Menstrual blood differs from human circulatory blood in terms of its content and its properties. Circulatory blood contains a higher concentration of proteins than in menstrual blood. The major protein is 'fibrinogen' which is a clotting factor in the plasma of blood. This cause menstrual blood to not clot as much as normal blood and is a major marker to differentiate the two.

85. Menstrual blood Test for menstrual blood Absence of fibrinogen is a feature that is tested in menstrual blood. Latex Agglutination technology is used for this purpose.

86. Menstrual blood Hormonal tests for FSH, LH etc. are also available.

87. Sweat Sweat or perspiration is the colorless fluid secreted by the sweat glands in the skin of humans. Sweat contains water, electrolytes, lactic acid and urea. Sweat glands may be eccrine (all over the body) or apocrine (situated in armpits) glands. Sweat is a thermoregulatory process. The water and salts that are secreted help to regulate temperature in the human body owing to evaporation. Sweat does not contain any cellular material.

88. Sweat

89. Sweat Tests for sweat Sweat does not have cellular fraction. Therefore unfit for DNA. Sweat is usually used to detect drug abuse. Metabolites which are specific for the suspected drug are tested. Special kits are available for each narcotic substance.

90. Milk Milk (Lactation) is a nutrient-rich liquid secreted by the mammary glands (breasts) of women to nourish their offspring (child). The chief function of milk secretion is to provide nutrition. It also provides immunity, emotional connection etc. Milk contains nutrients, proteins and lactose. The first milk produced after childbirth is known as colostrum and contains antibodies in addition to the above . Human milk and animal milk differs in certain means – PUFA lipoproteins and Vitamin D are present in human milk and absent in animal milk.

91. Milk Tests for milk Lactose is a target molecule – Reagent test kits Vitamin D, LDLs can also be looked for using biochemical analysis.

92. Vomitus Vomitus is another biological specimen rarely encountered in cases. They contain mucus, free acid ( HCl ), endothelial and epithelial cells from the gut lining, undigested food material etc. They can help us understand the last meal of the person, any toxic or metabolic overdose etc.

93. Vomitus Test for vomitus For mucus – 33% acetic acid is added to stain – Opalescence disappears if it is a vomitus stain. For HCl – 10% phloroglucinol is added followed by 10% Vanillin – Brilliant red color indicates presence of free HCl .

94. Fetal blood Fetal blood is another occasional evidence encountered in India. The social evil of female infanticide, selective gender bias etc. ensures cases of this nature. When aborted material, infant bodies etc. are encountered, the blood of the fetal material becomes incriminatory evidence. Fetal blood can be differentiated from adult blood and this can help prove the corpus deliciti and motive . Adult blood has 2 alpha & 2 Beta subunits of hemoglobin, whereas fetal blood contains 2 alpha and 2 gamma subunits of hemoglobin.

95. Fetal blood Tests for fetal blood The test is known as APT test or Alkali denaturation test. Blood cells are first lysed. SO distilled water is added to the stain and the hemoglobin is extracted out of the RBCs. To the lysed Hemoglobin, 1%NaOH is added and left to stand in room temperature. Fetal hemglobin will stay pink, adult hemoglobin will turn yellow-brown,

96. Hair Hair is another biological specimen which is regularly encountered as evidence. We will deal with hair in a following unit in the paper.

97. Nail Nail, nail clippings, nail scrapings are another set of biological evidence. These are regularly encountered in cases of murder, sexual abuse, occupational crimes etc.

98. Nail Tests for nail Microscopy of nail scrapings is performed. The contents will usually be dirt. Blood or seminal stains can also be detected in specific cases. Hair could be caught in between nails.

99. Tissue & Bones Tissues and bones of victims are seen in murder cases and other violence related cases. they help to prove the events that ensued during the crime.

100. Tissue & Bones Tests for tissue & Bones Tissue needs to be either processed on urgent basis or needs to be preserved in formalin. But formalin preservation is discouraged for blood testing / poison testing. This must be kept in mind for choosing the right preservation technique. Tissue is studies microscopically after making thin cross section using microtome, to identify the organ from which the tissue has come from. Bones need to be dried and preserved in crystal salt and sent to laboratory. Bones can help in DNA analysis, calcium levels and extent of osteoporosis etc.

101. Vaginal secretions Vaginal secretions or fluid is a rare biological evidence. In cases of rape it can be used as an alternate means of proving the crime.

102. Vaginal secretions Tests for vaginal secretions Vaginal secretions appear stiff on feeling when they are on clothing. Under UV light they show greenish yellow fluorescence. Vaginal secretions also contain acid phosphatase. Lugol’s stain is performed. Epithelial cells (Squamous) are typical findings in vaginal secretions. They appear chocolate brown / tan color on staining.

103. Collection of biological evidences Biological evidences have the chance of decomposition and rotting. This makes collection & preservation of biological material significant. Many cases get wasted due to poor packaging of biological evidences.

104. Collection of blood Blood evidence is the most common biological evidence in criminal cases. It is also one of the most evidentiary- ly valuable evidences. When not collected properly biological evidences tend to putrefy and go wasted. But there is also a practical difficulty of transportation and storage of biological evidences before analysis which makes it even more trickier. Blood evidence is encountered as both liquid blood and dried blood stains. Sometimes blood stained articles may also be recovered in crimes as evidence. Therefore, for each of these types of blood evidence, a separate collection and packaging protocol must be taken up.

105. Collection of blood Liquid blood evidence Liquid blood is commonly seen in murder scenes. Blood has a tendency to clot even after it has been bled from a person. Therefore, blood starts coagulating when spilt. This coagulation takes at least half an hour once it happens outside the body. During coagulation, the fibrinogen and pother clotting factors coagulate the blood into a rigid coagulum. Fibrinogen gets converted to fibrin and clumps up the cells and plasma of blood forming hard masses of blood.

106. Collection of blood The ideal form of collection of liquid blood would involve collection of liquid blood as it is and preserving using preservatives such as sodium citrate or sodium fluoride along with refrigeration until it is analyzed. But in the forensic point of view, the time taken for evidences to reach the FSLs is too long and ideal preservation is not possible. Therefore, it has to be preserved in different means. Liquid blood is best collected onto fibers of cotton threads. The layman understanding of collection of blood is to soak it in cotton wool or filter paper. But both those practices are forensically damaging as cellulose in paper can interfere with blood grouping results, and the cotton wool can act as artifacts during microscopy and make the job of analysis difficult. The best preservation medium for blood stains is threads of cotton. The ideal medium which has threads of cotton is ‘gauze cloth’. Therefore, clean sterile gauze cloth is used to collect liquid blood by soaking on it. And later the wet blood must be dried under shade (not in direct sunlight). Direct sunlight would destroy the antigenic nature of blood group antigens. Therefore, it has to be dried in the shade . The air dried, blood stained gauze cloth can be placed in a paper envelope and transported to the FSL.

107. Collection of blood

108. Collection of blood Dried blood stains on immovable property Blood does not stay liquid for long time. It first clots and then dries when left uncollected for a long time. Sometimes if the volume of blood is less that has fallen on a wall or weapon etc. it tends to dry up quickly.

109. Collection of blood Such dried blood stains cannot be soaked up on gauze cloth. Therefore, the dried stain must be reconstituted to make it wet. Water could be the first choice which comes to our mind. But water lyses the RBCs and makes the evidence wasted. This happens because human blood has an osmotic potential of around 7.8. At this osmotic potential the blood cells survive normally. But water’s osmotic potential ranges between 0-4. When water comes in contact with blood, the blood cells shrink and disintegrate – a process known as lysis. Hemoglobin is released out of the RBCs when they shrink and the entire sample goes wasted.

110. Collection of blood

111. Collection of blood Therefore, a liquid with equal osmotic potential as that of plasma is required to reconstitute the dried blood stain. Normal Saline (0.85% NaCl ) has been found to have the same osmotic potential of plasma. Therefore, few drops of normal saline may be poured on to the dried blood stains and once enough liquid blood is available it can be soaked on to gauze cloth and air dried.

112. Collection of blood Dried blood stains on moveable property When dried blood stains are encountered in moveable property like a weapon or a piece of brick or clothes, the entire blood stained article must be shifted to the FSL. The item must be packed with loose paper and forwarded to the FSL. It must be ascertained whether the blood is dry, if not the article must be air-dried before packing.

113. Collection of blood Blood stains mixed with other evidences Sometimes blood may be mixed with soil, or vegetative matter or pieces of glass etc. At such times, the blood stains must be collected along with the other items, air dried and then packed into paper envelopes or druggist folds and forwarded to FSL.

114. Collection of blood Blood evidence for DNA analysis Blood evidence for DNA analysis requires a particular mode of preservation. While sterile gauze cloth is also suitable, ther eis another medium more suited for DNA extraction. The FTA (Flinders Technology Associates) cards are cotton-based, cellulose paper containing chemicals that burst cells, denature proteins and protect DNA, leaving a sample suitable for molecular identification without the risk of contamination. All the user needs to do is add a drop of biological specimen - blood, semen etc. on the FTA card and preserve the card in paper envelopes.

115. Collection of blood Blood patterns Blood patterns is another piece of evidence when we regard blood. While the analysis of blood patterns will be dealt with later, it is good to understand that blood when it bleeds from a human body forms certain patterns which can help understand the chain of events during the crime. Therefore, blood stain patterns are significant findings in crime scenes. Photographs, tracing and sketching is usually the ways in which blood stain patterns can be documented.

116. Collection of semen Semen evidence is encountered in sexual offences like rape and unnatural sexual offences. Semen is the secretion of the male reproductive system and it carries the spermatozoa or male gamete. Semen is a whitish, curdy liquid usually secreted in very low quantities approximately 3.5ml during a single ejaculation. Semen dries up and forms a starchy stain on the material it falls on.

117. Collection of semen We may encounter semen as liquid semen during control collection from a suspect, or dried seminal stains on clothes, or dried semen matted on hair (usually genital hair). Occasionally semen lodged in the victim’s vagina may also have to be collected as swabs.

118. Collection of semen Liquid semen Liquid semen is collected form suspects in order to compare with the other evidences in rape cases. The suspect will be asked to collect semen in a clean sterile glass bottle. The sample will be measured for volume, later it will be allowed to liquefy in room temperature. Liquefaction takes about 20 minutes and changes the gel-like liquid to a liquid form. It is essential because demonstration of spermatozoa in gel semen is impossible. Once liquefied the semen will be smeared on microscopic glass slides and labelled and air dried and placed in paper envelopes and sent to the FSL. The remaining liquefied semen will be soaked on sterile clean gauze cloth, air dried and sent to the FSL.

119. Collection of semen Dried seminal stain Semen dries when it falls on clothes and forms a starchy stain. It is rough to touch and can be usually made out with touch. Dried seminal stains fluoresce under UV light. This is a useful means of identifying dried seminal stains as sometimes it may be difficult to pick up or may be confusing to decide between seminal stains and other biological stains . Once the dried seminal stain is located, the entire cloth can be forwarded to FSL. The cloth must be folded in such a way as to avoid the seminal stain from being transferred to other regions of the cloth. Therefore, the cloth may be folded in such a way as to keep the seminal stain on top of the cloth. It is a good practice to mark the stains also using marker pens before forwarding to the FSL.

120. Collection of semen Dried semen matted on hair Semen when it falls on hair, it tends to matt the hair. Hair strands stick onto each other and forms a matte. This matted hair is encountered in genital hair in rape cases etc. Such matted hair may be cut using scissors, air dried and packed in druggist folds and placed in envelopes and forwarded to FSL.

121. Collection of semen Semen lodged in body cavities Semen can also be suspected to be lodged in the victim’s vaginal tract in cases of rape. In other unnatural sexual offenses semen can be suspected of being lodged in other body cavities. It is a significant finding to prove the crime. Therefor it need to be collected. Swabs are the best way to collect such specimen. Sterile clean cotton swabs are inserted and the sample is taken and labelled with the name of the body cavity. Vaginal swab, vulval swabs, oral swabs, anal swabs etc. are collected for detecting presence of semen.

122. Collection of semen Semen for DNA analysis FTA cards are used similar to what we discussed in collection of blood.

123. Microbiology Microbiology is the branch of science that deals with microscopic organisms and their interaction with other microscopic and macroscopic organisms. Microbiology occupies a central role in medical science as the microbes cause a whole lot of infections. Microbes can be bacteria, viruses, fungi or even parasites. Therefore, microbiology is subdivided into bacteriology, virology, mycology and parasitology respectively. Microorganisms are tiny microscopic organisms that are too small to be seen with naked eyes and thus, can only be seen with a microscope. Microbiology also deals with the structure, function, classification of such organisms, along with exploiting and controlling their activities. The concept of microbiology began with the discovery of the microscope by Anton Von Leeuwenhoek.

124. Microbiology A portion of microbes cause diseases. On the one hand, microbes are used for their unique features which allow the production of antibiotics, amino acids, hormones, and other therapeutic compounds, and also the production of food and food-related products. Microorganisms are also involved in the decomposition of components such as lignocellulosic biomass for second-generation ethanol or biogas. Similarly, certain genetic features and biochemical abilities of microorganisms make them dangerous for industry (food spoilage) as well as human health. Microbiology, initially, was only associated with pathogenic microorganisms that result in different forms of diseases in different groups of living beings.

125. Microbiology With the establishment of microbiology as a discipline, the application of microorganisms in different areas has also increased. The use of microorganisms in food and pharmaceuticals has given rise to the branching of microbiology into further disciplines and studies. Thus, over the years, the branch has been classified into further groups like agriculture microbiology, food microbiology, pharmaceutical microbiology, systemic microbiology, etc. We will quickly understand the basic microbiology necessary to understand forensic microbiology.

126. Microbiology Bacteriology Mycology Virology Parasitology

127. Bacteriology Bacteriology is a branch or discipline of science that studies different characteristics of bacteria and their association with other organisms or disciplines. Every human gut contains ‘normal flora’ – necessary bacteria which helps digestion. There are also disease causing bacteria – pathogens. Bacteria are microscopic and can be multiple shaped. Based on their shape and chemical nature, they are classified into multiple types.

128. Bacteriology Coccus: Round shaped bacteria. Bacillus: Road shaped bacteria. The common coccus bacteria are: Staphylococcus sp.: Cluster of round shaped bacteria. They cause skin infections Streptococcus sp.: Chain of round shaped bacteria. They cause throat infections. The common bacillus bacteria are: Salmonella sp.: They cause typhoid. Escherichia coli: Cause dysentery. These are just a few examples. There are multiple types of bacteria existing in the present world. Bacteria can be reared in nutrient medium like agar plates and can be studied, reared and propagated in that means.

129. Bacteriology

130. Bacteriology

131. Bacteriology

132. Bacteriology Bacteria need to be reared in lab from our samples, to prove bacterial infections. To rear bacteria we use different mediums – liquid / solid

133. Bacteriology

134. Bacteriology

135. Bacteriology

136. Bacteriology

137. Bacteriology

138. Bacteriology

139. Bacteriology

140. Mycology Fungi range from mushrooms to yeast and other common household products. Forensically ergot sp. is a fungus which is a irritant poison. Medically fungal infections are high risk.

141. Mycology

142. Mycology

143. Mycology

144. Mycology

145. Mycology

146. Virology Study of viruses. Classification is the major part of virology. DNA virus – smallpox; chickenpox RNA virus – common cold, fever, flu Reverse Transcribing / Retro – virus – HIV

147. Virology

148. Virology

149. Virology

150. Parasitology Study of the relationship between host, parasite and vector. Malaria, filariasis (elephantiasis), amebiasis .

151. Parasitology

152. Parasitology

153. Parasitology Malaria, Filaria , Head louse, ticks, intestinal worms etc.

154. Parasitology Study of the relationship between host, parasite and vector. Malaria, filariasis (elephantiasis), amebiasis .

155. Bioweapons The use of biological beings to create catastrophe. Biological agents like microbes can be very difficult to detect and can infect large populations in a very inexpensive manner. This practice has been happening through the ages.

156. Bioweapons MICROBE NATURE STABILITY REQUIRED DOSE Anthrax Bacteria - Bacilli Very stable 10,000 – 50,000 organisms Smallpox Virus Stable for 1 year 10 – 1000 organisms Ebola Virus Unstable 10 – 100 organisms Cholera Bacteria – Bacilli Unstable 10 – 500 organisms Brucella Bacteria – Bacilli Very stable 10 – 100 0organisms Mycoplasma Bacteria – Cocci Moderately stable 10 – 100 0organisms Corona Virus Virus Moderately stable 10 – 50 organisms

157. Bioweapons Must be contagious Must require very little dose – tasteless & odourless Must be difficult to detect Must be stable for transport Must be user-controllable

158. Review General Microbiology Bacteriology – Cocci & bacilli Mycology – Candida & Actinomyecetes Virology – DNA, RNA & retro viruses Parasitology – Worms & ticks

159. Bioweapons MICROBE NATURE STABILITY REQUIRED DOSE Anthrax Bacteria - Bacilli Very stable 10,000 – 50,000 organisms Smallpox Virus Stable for 1 year 10 – 1000 organisms Ebola Virus Unstable 10 – 100 organisms Cholera Bacteria – Bacilli Unstable 10 – 500 organisms Brucella Bacteria – Bacilli Very stable 10 – 100 0organisms Mycoplasma Bacteria – Cocci Moderately stable 10 – 100 0organisms Corona Virus Virus Moderately stable 10 – 50 organisms

160. Bioweapons Must be contagious Must require very little dose – tasteless & odourless Must be difficult to detect Must be stable for transport Must be user-controllable

161. Forensic Microbiology The field of forensic microbiology is fairly new and still evolving. With a threat of bioterror and biocrime , the rapid identification and subtyping of infectious agents is of upmost importance . Forensic gene typing is a variant of DNA fingerprinting. It helps identify the mutations that occur in viruses – easily appreciable in today’s covid times. Forensic microbiology works to detect origin, treatment plans etc. in all microbiological outbreaks.

162. Forensic Microbiology

163. Forensic Microbiology Soon after the 9/11 attacks, anonymous letters spread with deadly anthrax spores began arriving at media outlets and offices of different US leaders. Just by opening the envelope people were exposed to the deadly anthrax bacilli. 5 people died and 17 others were exposed within a day.

164. Forensic Microbiology

165. Forensic Microbiology Anthrax is a infectious bacillus (Bacillus anthracis ). It is found commonly in cattle and it causes severe pneumonic infection. Bacillus Anthracis is a rod-shaped bacterium arranged in long and branched chains.

166. Forensic Microbiology

167. Forensic Microbiology This created a massive confusion in the world arena. The lack of microbiological knowledge in forensic teams was exposed. Dependence on medical staff was compulsive, this resulted in the news pouring our with rumors and conspiracies in an already confused world.

168. Forensic Microbiology During the anthrax attack incident, the bacillus spores were collected and examined in microbiological labs. They were grown on blood agar plates. They grew as 2-3mm colonies with an edge like that of matted hair. The bacteria also formed hemolysis in the blood agar plates.

169. Forensic Microbiology

170. Forensic Microbiology A treatment plan was also quickly planned out using antibiotic susceptibility testing. There could be a cure to the damage, but it was delayed, and the damage was partly already done.

171. Forensic Microbiology Future bioweapons are at this moment getting prepared. Bioterrorism is the unlawful use or threatened use of microbes to produce death or disease in humans, animals or plants. The act is to create fear and intimidate governments or societies for political, religious or ideological goals .

172. Forensic Microbiology Future bioweapons are at this moment getting prepared. Bioterrorism is the unlawful use or threatened use of microbes to produce death or disease in humans, animals or plants. The act is to create fear and intimidate governments or societies for political, religious or ideological goals .

173. Forensic Microbiology Microbes are part of occasional crime. There have been a recorded history of many such crimes which include microbes as weapons of war. 1346 - Siege of Kaffa - plague 1763 - French and Indian war - Smallpox 1865 - WW I - German pogrom - Anthrax and glanders 1925 - Geneva protocol bans biological weapons 1935 - WW II - Japanese porgrom - Anthrax, plague, cholera and shigella

174. Forensic Microbiology 1946 - US anounces official bioweapons research unit 1969 - US stops the bioweapons research after a public uproar. 1972 - Biological weapons convention gathered. 1979 - Accidental release of Bacillus anthracis spores in USSR 1984 - Rajneesh cult - Salmonella typhi outbreak 1992 - Ricin attack Minessota 2001 - Anthrax release in the US. In all the above instances, slowly but steadily the world is witnessing an increase in the possibility of using a biological weapon. Biological weapons can be dangerous and threatening to the entire humankind.

175. Bioweapons - Classification Class A: Contagious with high death rate and health impact on public. Eg . Anthrax. Covid etc . Can cause high morbidity Transmission would be person to person Potential for a major public health impact Might cause public panic and social disruption

176. Bioweapons - Classification Class B: Moderately easy to spread. Causes some illness and death rates. Eg . Typhus fever . Moderately easy to disseminate Causes moderate morbidity and low mortality Surveillance may be reqd. post the attack for quite some time.

177. Bioweapons - Classification Class C: Easily available, can be easily produced and spread. Have poten Usually would be pathogens that would be engineered to cause little more damage to public health. Hantaviruses, tick-borne elephantiasis virus Multi-drug resistant tuberculosis etc. Can cause little panic.tial for less life loss. Eg . cholera.

178. Bioweapons - Spread Bio weapons can be easily spread. That’s what makes it significant. They can be easily spread by different delivery methods such as: Food / Water Air handling systems Aircraft sprayers Vehicle sprayers human vectors Hand sprayers Animal sprayers Mail

179. Bioweapons - Spread The ideal bioweapon would be contagious, virulent, robust, difficult to detect, drug-resistant and user-controllable. No naturally occurring microbe fulfils these conditions. Bioweapon terror involves in modifying existing microbes to fulfil these conditions.

180. Forensic Microbiology Forensic microbiology is a branch of forensic science which deals with microbes and their use in crime. It needs to understand the basics of microbiology in order to be able to identify the crime in cases such as bioweapon usage. Cases such as the spread of anthrax bacteria through mails and couriers in the US have generated a lot of interest in this field. Forensic Microbiology units have been set up in many parts of the world.  In India, only FSL, Delhi and CFSL, Hyderabad have dedicated Forensic Microbiology units as of now . GFSU has a Forensic Microbiology unit as well as a specialty course on Forensic Microbiology.