Académique Documents
Professionnel Documents
Culture Documents
Submitted by
Shourya Sharma
(Batch: 2014 - 2017)
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UNDERTAKING
I hereby declare that the matter embodied in this dissertation entitled Advance Techniques in
Forensic Science, submitted to the Department of Applied Chemistry, Amity School of Applied
Sciences, Amity University Gurgaon, Haryana, for the award of the degree of Bachelor of Science
Honours in Forensic Science is my original work under the guidance of Dr. Mohit Soni. Further,
I would like to state that the work is not based or reproduced from any existing work already done
by any other person for any other purpose and it has also not been submitted anywhere else at any
time.
Shourya Sharma
A51605914008
Department of Applied Chemistry
Amity University Gurgaon
Haryana
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Mr.Ravi Rathi
Teaching Associate
Co-supervisor
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ACKNOWLEDGEMENT
The success and final outcome of this training required a lot of guidance and
assistance from many people and I am extremely fortunate to have got this all
along the completion of my training. Whatever I have done is only due to such
guidance and assistance and I would not forget to thank them. I respect and thank
Dr. Tripti Bhatnagar, for giving me an opportunity to do the training in Codon
Biotech Pvt. Ltd and providing me all support and guidance which made me
complete the training on time.
I owe my profound gratitude to my Co supervisor, Mr. Ravi Rathi, who took
keen interest in my training and guided me all along.
I would also like to acknowledge Dr. Mohit Soni, Department of Forensic Science
for his continued support and knowledge throughout my internship. He continually
guided me during my internship and for sharing his expertise, and sincere and
valuable guidance and encouragement to me.
I would also like to thank my family for always being there for me and giving me
moral support throughout my life.
Shourya Sharma
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CONTENTS
Page No.
1. Acknowledgement
2. Summary
3. Review of Literature
17
4. Experimental Section
19
Microbial Forensics
19
21
23
27
28
30
DNA Fingerprinting
31
Blood Grouping
32
Haemoglobin Estimation
34
RBC Counting
36
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37
39
41
44
46
47
49
51
Paper Chromatography
52
Gas Chromatography
54
5. References
57
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SUMMARY
Summer Training at Codon Biotech Pvt. Ltd was four weeks long in which they thought us various
advanced techniques that are used in Forensic Science. Codon Biotech is a multifaceted company
involved in Biotechnology related research, training and manufacturing, incorporated in the year
2007 in India. It is located in Noida. Its four research strands comprises of: the Agriculture Biotech
Sector, Environmental Biotech Sector, Industrial Biotech Sector and Biotechnological Kits &
Aromatic oil Manufacturing Sector. Codon Biotech aims to motivate and promote students by
providing the excellent hands on practical experience to meet the rapidly evolving opportunities
& challenges in the field of biotechnology.[1]. The topics covered at Codon Biotech were-
1. MICROBIAL FORENSICS:
Microbial forensics has been defined as a scientific discipline dedicated to analyzing evidence
from a bioterrorism act, biocrime, or inadvertent microorganism/toxin release for attribution
purposes. This emerging discipline is still in the early stages of development and faces substantial
scientific challenges to provide a robust suite of technologies for identifying the source of a
biological threat agent and attributing a biothreat act to a particular person or group. The unlawful
use of biological agents poses substantial dangers to individuals, public health, the environment,
the economies of nations, and global peace. It also is likely that scientific, political, and mediabased controversy will surround any investigation of the alleged use of a biological agent, and can
be expected to affect significantly the role that scientific information or evidence can play. For
these reasons, building awareness of and capacity in microbial forensics can assist in our
understanding of what may have occurred during a biothreat event, and international collaborations
that engage the broader scientific and policy-making communities are likely to strengthen our
microbial forensics capabilities. One goal would be to create a shared technical understanding of
the possibilitiesand limitationsof the scientific bases for microbial forensics analysis.[1]
Staphylococcus spp
Streptococcus spp
Pneumococcus spp
B Diphtheria
Clostridium spp
B Anthracis
Lactobacillus spp
Gonococcus spp
Meningococcus spp
Pasteurella pestis
Brucella spp
Salmonella spp
Vibriio cholera
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Crystal violet dissociates in aqueous solutions into CV and chloride ions. These ions penetrate
through the cell wall and cell membrane of both gram negative and positive cells. The CV ion
interacts with negatively charged components of bacterial cells and stains the cell purple.
Iodine interacts CV and forms large complexes.
Iodine is often referred to as a Mordant, but is a trapping agent that prevents the removal of the
CV-I complex and, therefore, colors the cell.
When alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram
negative cell loses its outer lipopolysaccharide membrane and the inner peptidoglycan layer is left
exposed. The CV-I complexes are washed from gram negative cell along with the outer membrane.
In contrast, a gram positive cell becomes dehydrated from an ethanol treatment. The large CV-I
complexes become trapped within the gram positive cell due to the multilayered nature of its
peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet
stain is removed from both gram positive and gram negative cells if the decolorizing agent is left
on too long.
After decolorization, the gram positive cell remains purple and the gram negative cell loses its
purple color. Counter stain, which is usually positively charged safranin is applied last to give
decolorized gram negative bacteria a pink or red color.
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matrix allows small molecules to move quickly through the gel, whereas the larger molecules are
hindered by the matrix, causing them to migrate slower. The addition of an electric current to the
gel causes the molecules to move in one direction, towards the positive end of the gel. DNA
molecules are negatively charged and therefore migrate through the Agarose gel matrix to the
positive terminal at the bottom of the gel.
6. PLANT GENOMIC DNA:
A typical fungal cell has complex protoplasm containing micro vesicles, microtubule, ribosome,
mitochondria, Golgi apparatus, nuclei and a double membrane cytoplasmic reticulum. The nucleus
is enclosed by a defined nuclear membrane with its entire DNA and has a nucleolus rich in RNA.
The amount of DNA in a single fungal cell is about 4-10 times that of bacterium but
only1/1000times that of a plant or animal cell. A membrane known as plasmalemma, composed
of glycoprotein, lipids and ergosterol encloses the entire protoplasm. The compound ergosterol is
unique to fungi contrasting with mammals which have cholesterol. Outside the plasmalemma, a
multilayered cell wall is present. The wall which constitutes 90% of the dry weight of the fungus
is complex containing chitin, a polymer of N-acetyl glucosamine as its structural base. The chitin
is layered with mannas, glucans and other complex polysaccharides amidst polypeptides. In
filamentous fungi, chitin synthetase. Some fungi have a capsular polysaccharide outside the cell
wall which serves to isolate fungi from their environment but at the same time acts as liason
between the outside environment and cell contents.
7. DNA FINGERPRINTING:
DNA fingerprinting, also called DNA typing, in genetics, method of isolating and making images of
sequences of DNA (deoxyribonucleic acid).The technique was developed in 1984 by the British
geneticist Alec Jeffreys, after he noticed the existence of certain sequences
of DNA (called minisatellites) that do not contribute to the function of a gene but are repeated
within the gene and in other genes of a DNA sample. Jeffreys also determined that each organism
has a unique pattern of these minisatellites, the only exception being multiple individuals from a
single zygote (e.g., identical twins). Unlike a conventional fingerprint that occurs only on the
fingertips and can be altered by surgery, a DNA fingerprint is the same for every tissue, and organ
of a person. It cannot be altered by any known treatment.
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The alternative names of this test are: Cross matching, Rh typing, and ABO blood typing. Blood
is drawn from a vein, usually from the inside of the elbow or the back of the hand. The puncture
site is cleaned with a germ killing product.
9. HAEMOGLOBIN ESTIMATION:
Haemoglobin (also abbreviated Hb or Hgb) is the iron containing oxygen-transport metalloprotein
in the red blood cells of vertebrates, and the tissues of some invertebrates. A haemoglobin molecule
is composed of a protein group, known as globin, and four heme groups, each associated with an
iron atom. Haemoglobin in blood is what transport oxygen from lungs or gills to the rest of the
body (i.e. the tissues) where it releases the oxygen for the cell use. In mammals, protein makes up
about 97% of the blood cells dry content, and around 35%of the total content (including water).
Haemoglobin has an oxygen binding capacity between 1.36 and 1.37 ml O2 per gram of
haemoglobin, which increases the total blood oxygen capacity seventy fold.
H2O + O2
catalase
When this reaction occurs, the oxygen gas is released as bubbles. The catalase enzyme performs
an important function to living organisms because hydrogen peroxide is very toxic to living cells.
Other organisms, including plants and somebacteria, also make catalase. If you place a few drops
of hydrogen peroxide on a substance that contains catalase, it will bubble profusely. These
substances that bubble with the addition of hydrogen peroxide are said to test positive for catalase.
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Drug toxicology tests are most commonly performed on urine, since most drugs and their
breakdown products are excreted in the urine in higher concentrations than in the blood, and
because urine toxicology tests are often inexpensive and quick. Alcohol toxicology tests are
routinely performed on blood and breathe as well as urine. Tobacco smoking is considered to
increase the risk of a variety of cancers as well as cardiovascular disease .Nicotine is the most
abundant alkaloid found in tobacco and responsible for the addictive properties of cigarettes and
can be easily detected in urine, plasma, and saliva. Urinary cotinine is widely used as a biomarker
due to its higher concentration in the urine matrix as compared to other matrices, resulting
inaccurate detection in samples. Several different analytical methods have been described for
determination of urinary nicotine and cotinine, including colorimetry and Thin Layer
Chromatography. Thin layer chromatography can be used to monitor the progress of a reaction,
identify compounds present in a given mixture, and determine the purity of a substance. Specific
examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides
or insecticides in food and water, analyzing the dye composition of fibers in forensics, as saying
the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their
constituents.
15. DETECTION OF INK BY PAPER CHROMATOGRAPHY:
Paper chromatography is an analytical method technique for separating and identifying mixtures
that are or can be coloured, especially pigments. This technique has important use in forensic
sciences. Paper chromatography can separate minute amounts of substances. This makes it very
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useful for forensics investigators. Drugs from narcotics to aspirin can be identified in urine and
blood by using chromatography. Detectives can analyse a sample of paint to find out the car it
came from, including the model and year it was made. Ink can be separated into its components to
help identify the pen used for ransom notes and forgeries, as well as the type of ink used for
counterfeiting.
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or in the soil. The diatoms are identified by their morphological characteristics. The siliceous
exoskeleton imparts uniqueness to their structure. Diatoms are extracted from the various body
tissues by various methods of digestion and centrifugation. Diatom test is based on a theory that
normally people do not have significant number of diatoms in some important organs like kidneys,
liver, brain and bone marrow etc. but if a person drowns in water containing diatoms, the diatoms
in that water will reach the lungs and some of them will penetrate the alveolar wall.
Instrumental components:
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon
and carbon dioxide. The choice of carrier gas is of often dependent upon the type of detector which
is used.
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For optimum column efficiency, the sample should not be too large, and should be introduced onto
the column as a plug of vapour slow injection of large samples causes band broadening and
loss of resolution. The most common injection method is where a micro syringe is used to inject
sample through a rubber septum into a flash vaporiser port at the head of the column. The
temperature of the sample port is usually about 50C higher than the boiling point of the least
volatile component of the sample.
Columns
There are two general types of column, packed and capillary (also known as open tubular). Packed
column contain a finely divided, insert, solid support material (commonly based on diatomaceous
earth) coated with liquid stationary phase. Most packed columns are 1.5-10 m in length and have
an internal diameter of 2-4 mm.
Capillary columns have an internal diameter of a few tenths of a millimetre. They can be one of
two types:
Wall coated columns consist of a capillary tube whose walls are coated with liquid stationary
phase.
In support-coated columns, the inner wall of the capillary is lined with a thin layer of support
material such as diatomaceous earth, onto which the stationary phase has been adsorbed.
SCOT columns are generally less efficient than WCOT columns. Both types of capillary column
are more efficient than packed columns.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree. The
optimum column temperature is dependent upon the boiling point of the sample. As a rule of
thumb, a temperature slightly above the average boiling point of the sample results in an elution
time of 2-30mins. Minimal temperature gives good resolution, but increase elution times. If a
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sample has a wide boiling range, then temperature programming can be useful. The column
temperature is increased (either continuously or in steps) as separation proceeds.
Detectors
There are many detectors which can be used in gas chromatography. Different detectors will give
different types of selectivity. For example:
The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds
burning in the flame produce ions and electrons which can conduct electricity through the flame.
A large electrical potential is applied at the burner tip, and a collector electrode is located above
the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDS
are mass sensitive rather than concentration sensitive; this gives the advantage that changes in
mobile phase flow rate do not affect the detectors response. The FID is a useful general detector
for the analysis of organic compounds; it has high sensitivity, a large linear response range, and
low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.
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REVIEW OF LITERATURE
CASE STUDIES:
1. Fiber evidence in the Wayne Williams case:
Before Wayne Williams became a suspect, the Georgia State Crime Laboratory located a number
of yellowish-green nylon fibers and some violet acetate fibers on the bodies of victims murdered
in the Atlanta area. The fibers appeared to have a common source. Shortly after Williams became
a suspect, the investigators determined the source of the fibers' manufacture. The next step was to
determine how much of that carpeting has been sold in the Atlanta area. Further investigation
traced the fiber to a limited amount of carpeting manufactured by West Point Pepperell. To convey
the unusual nature of the Williams carpet, investigators developed a numerical probability from
that company's data. They determined that the chance of randomly finding a housing unit with this
type of carpet was 1 in 7,792. To any experienced forensic fiber examiner, the fiber evidence
linking Williams to the murder victims was overwhelming. The prosecution undertook to educate
the jury about fiber evidence, using over 40 charts and over 350 photographs. In discussing the
significance of an association based on textile fibers, it emphasized that the more uncommon the
fibers, the stronger the association. The association becomes even stronger when multiple fiber
matches become the basis of the association, which was true in the Williams case. Transferred
fibers do not stay with a person long, so that the fibers on a murder victim are most likely from the
scene of the crime. Expert witnesses testified that it was highly unlikely that any environment other
than that present in Wayne Williams' house and car could have produced the combination of fibers
and hairs found on the victims, especially when there were so many varied origins. Additional
evidence linked 10 other victims to 28 different fiber types, only 1 of which was common. Nine
footnotes and three charts are included.[2]
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was made public, scholars questioned the validity of the map because of its surprisingly accurate
depiction of Greenland, which was drawn as an island on the Vinland Map. Ships at the time were
not capable of traversing the arctic waters that surrounded Greenland to the north. To add to the
speculation, British Museum researchers had previously determined that different inks were used
on the Vinland Map and within the Tarter Relation book. They also noted that under ultraviolet
light the ink behaved unlike any other medieval ink they had encountered in the past. yale
university sent the Vinland map to McCrone Associates in 1972 for a comprehensive analysis of
the inks utilized. As a leader in microanalysis, McCrone Associates had the technology and the
experience to conduct an in-depth investigation.Upon initial viewing with a stereomicroscope,
Anna Teetsov, Senior Research Microscopist at McCrone Associates, determined that the map was
double-inked. Maps of authentic medieval origin develop a yellow stain along the ink lines from
the natural leaching of the ink into the parchment fibers over time. According to Teetsov, yellow
ink was used to simulate the stain on the Vinland Map and then black ink was drawn over it,
indicating that the parchment surface was altered to make it appear older than it actually was. Ms.
Teetsov was able to carefully lift 29 ink samples from the map for further analysis. The team then
examined the samples using an array of ultramicroanalysis techniques such as polarized light
microscopy (PLM), electron microprobe analysis (EMA), X-ray diffraction (XRD) and
transmission electron microscopy (TEM) to determine the chemical makeup and crystalline
structure of the ink. The critical microanalysis studies were performed on the yellow stain found
on the Vinland Map. These results of these tests showed that the yellow ink stain, visualized under
the stereomicroscope, contained titanium oxide (TiO2) pigment in its 0.3 micrometer synthetically
crystallized form of anatase. This form of TiO2 was only available in ink after its patent in 1917.
According to Walter C. McCrone, These particles are unique and impossible to have been
prepared in 1440, 300 years before titanium was discovered and nearly 500 years before the
chemical synthesis of pigment titanium dioxide was developed and, with great effort and expense,
perfected to yield 0.3 micrometer anatase pigment.
McCrone Associates concluded that the Vinland Map was a clever forgery using ink that was not
available until the early 20th century. Their findings support the claims made by the British
Museum that the ink was not of medieval origin.[4]
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EXPERIMENTAL SECTION
MICROBIAL FORENSICS
AIM- To prepare Nutrient Agar and Nutrient Broth.
PROCEDUREComposition of nutrient broth: NaCl-8gm
NaCl-0.8gm
Peptone-5gm
Peptone-0.5gm
Beef extract-3gm
Beef extract-0.3gm
D/W-1000ml
D/W-100ml
pH-7
pH-7
Preparation of nutrient broth:1. The chemical ingredients of the media were weighed and transferred to a beaker/flask
containing distilled water.
2. The ingredients were properly dissolved.
3. The pH of the medium were measured and adjusted to the required pH using HCL or NaOH
depending on the pH.
4. After that the mouth of the flask was caped using a cotton plug and tightly covered with
aluminium foil.
5. The flask was then autoclaved with the ingredients at 121C for 30 minutes.
NaCl-0.8gm
Peptone-5gm
Peptone-0.5gm
Beef extract-3gm
Beef extract-0.3gm
Agar-20 gm
Agar-20 gm
D/W-1000ml
D/W-100ml
pH-7
pH-7
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Preparation of Nutrient Agar Media:1. The chemical ingredients of the media were weighed and transferred to a beaker/flask
containing distilled water.
2. The ingredients were properly dissolved.
3. The pH of the medium were measured and adjusted to the required pH using HCL or NaOH
depending on the pH.
4. After that the mouth of the flask was caped using a cotton plug and tightly covered with
aluminium foil.
5. The flask was then autoclaved with the ingredients at 121C for 30 minutes.
6. The material was then transferred in petriplate and test tubes inside laminar flow cabinets.
7. The tubes were then pluged and petriplates were covered.
8. The test tubes and petriplates were left until they solidified.
RESULT AND DISCUSSIONThe whole experimental set was started with preparation of media for the growth of Bacteria.
Nutrient agar media was thus prepared. Autoclaving was done to sterilize the media at 15 psi for
15 minutes. The media once autoclaved was stored at 40C for future use.
FORENSIC SIGNIFICANCEMicrobial forensics is a relatively new field that can help in solving cases such as: bioterrorism
attacks. medical negligence. outbreaks of foodborne diseases. Microbial forensic data must hold
up not only to the scrutiny of scientists in the health care community, but also to the scrutiny of
P a g e | 21
judges and juries. If a biocrime is committed microbial forensic evidence can be sought, collected,
and characterized to help investigators identify the perpetrator(s) and exclude innocent suspects.[5]
SCOPEDisciplines related to microbial forensics are evolving rapidly. This evolution includes technology,
analytical capabilities, and equally as important education and training. Microbial forensics
necessarily cover a broad range of topics such as microbiology, biotechnology, epidemiology,
evolution, statistics, infectious diseases, bioterrorism, etc. More importantly, the ultimate goal of
attribution is identification of the persons who committed the bioterrorist act or biocrime,
intentionally or inadvertently. In addition to microbiological analytical tools, traditional forensic
analyses, such as human DNA analysis, dermatoglyphic patterns, analytical chemistry, tool marks,
and other techniques will be used to analyze a bioterrorist event or biocrime evidence.[6]
Bacterial smear was prepared on bacteria and it was then heat fix and air dried.
On a rack, flooded with filtered crystal violet for 1 min.
It was washed briefly in water to remove excess crystal violet.
Flooded with Grams iodine for 1 min.
Again washed briefly with water without letting it to dry out.
It was then decolorized with acetone until the moving dye front had passed the lower edge
of the section.
7. Washed immediately in tap water.
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RESULT AND DISCUSSIONGram staining - Gram staining was done & the stained bacterial smears were visualized under
microscope and differentiate gram positive and gram negative bacteria.
CONCLUSIONGram-positive bacteria are bacteria that give a positive result in the Gram stain test. Grampositive bacteria take up the crystal violet stain used in the test, and then appear to be purplecoloured when seen through a microscope.
Gram-negative bacteria are a group of bacteria that do not retain the crystal violet stain used in
the Gram staining method of bacterial differentiation and then appear to be pink coloured when
seen through a microscope.
FORENSIC SIGNIFICANCEA gram stain is a type of microbiology or laboratory test that determines whether bacteria are
present. It also determines whether bacteria are gram negative or gram positive. The difference
between gram negative and gram positive bacteria can be important when determining appropriate
treatment for an infection. Gram stains are performed on various types of specimens including
blood, tissue, stool, and sputum. In some instances, it isnt clear whether an infection is bacterial,
fungal, or viral. And these types of infections may be treated very differently. In addition, different
types of bacteria may require different treatments. A gram stain lets physicians determine whether
bacteria are causing an infection and what type of bacteria is present. Thats why in cases of food
poisoning or if someone used bacteria as their weapon, so the particular type of bacteria can be
identified on the basis of gram staning.[7]
SCOPEGram stain is probably one of the most commonly used staining procedures used in the field of
microbiology. Gram staining technique is used for staining bacteria, yeasts and aerobic
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actinomycetes. This Standard Operating Procedure is applicable to all technical staff in the
microbiology section that have been trained in performing this procedure and are competent and
authorized to perform this procedure.[8].The classification of gram negative and gram positive
organisms is the essential first step in deciding the nature of an infection and the
appropriate antibiotic coverage for an infection before a culture and sensitivity is available. It is
the most common stain performed precisely because it is the most useful.[9]
Tryptone
Nacl
CaCl2
Distilled water
10gm
5gm
1gm
1000ml
PROCEDURE1. This mixture was autoclaved for 15 min at 15 psi and 5 ml was poured in autoclaved glass
tubes with cotton plugging.
2. Inoculated with the required culture and incubated at 370C for 48 hours.
3. After 48 hours, 500 micro liter of Kovaks reagent was added to the tube and the formation
of red color ring was observed on the surface of the broth. This showed that the indole
test is positive.
7gm
5gm
5gm
1000 ml
PROCEDURE1. This mixture was autoclaved for 15 min at 15 psi and 5 ml was poured in autoclaved glass
tubes with cotton plugging.
2. For MR VP separate tubes were poured for same bacterial cultural.
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1 gm
1 gm
2 gm
5 gm
0.2 gm
15 gm
0.8 gm
1000 ml
PROCEDURE1. The mixture was autoclaved for 15 minutes at 15 psi and 5 ml was poured in autoclave
glass tubes with cotton plugging and by making slants.
2. Inoculated with required culture and incubated at 370 C for 48 hours.
3. The color of agar slant was observed. The color changed to Prussian blue. This showed
that citrate test is positive.
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BACTERIAS
Ecoli
Enterobacter
Bacillus
Klebsiella
MR
VP
v/+/-
TEST
INDOLE
CITRATE
P a g e | 26
agarhas a pH of 6.9 so it is an intermediate green colour .Growth of bacteria in the media leads to
development of a Prussian blue colour (positive citrate).
Fig5:- Tube1 MR test, Tube2- Indole test, Tube3- VP test, Tube4- Citrate
test
CONCLUSIONOn the basis of above observation the bacteria present in the food sample was Enterobacter.
FORENSIC SIGNIFICANCEThe IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae). These are the
Indole test (tryptone broth), the Methyl Red and Voges-Proskauer tests (MRVP broth) and the
Citrate test (Citrate agar slants). These 4 IMViC tests constitute, perhaps, the most critical tests
used for identification of bacteria after the gram stain. The test results from these 6 tests should
carry more weight than almost any other tests, certainly higher priority than sugar results since
they are more stable reactions. With the help of IMViC tests, a forensic scientist can determine the
organisms causing the food poisoning or in case of an unknown disease, to find out the infectious
organisms and to identify the spoilage organisms which were intentionally added to the food or
water to detect the criminal.
P a g e | 27
BACTERIAL GENOMIC DNA ISOLATIONAIM- To isolate the bacterial genomic DNA from the given bacterial culture.
PROCEDURE ( alkaline lysis method)1. 2ml of bacterial culture was taken in a tube and centrifuged for 10 minutes, the
supernatant was discarded and then 0.2ml of cell suspension buffer was added to the
tube.
2. The tube was inverted several times to slowlt mix it.
3. 0.2ml of cell lysis buffer was added to the tube and mixed.
4. Then 0.2ml of DNA salt solution (Neutralization solution) was added to the tube and
mixed by inverting several times.
5. The tube was centrifuged for 10 minutes and the supernatant was collected carefully in
the new eppandorf tube.
6. 0.8ml DNA precipitating solution (100% alcohol) was added and then slowly inverted
the tube several times to mix it.
7. The DNA was pelleted by centrifuging at high speed for at least 10 minutes.
8. The supernatant was removed and pellet was washed with 0.5ml of 70% ethanol and
centrifuged at high speed for at least 10 minutes.
9. The ethanol was discarded then and the tube was kept open at room temperature until
complete drying.
10. The pellet was suspended in TE buffer to store at -20 degree C.
RESULT AND DISCUSSIONBacterial culture that was grown in Agar media was used for DNA Isolation. This method uses
DNA Salt Solution/Neutralisation Solution; hence, it is called Alkaline Lysis Method. Another
method uses PCI (Phenol:Cholroform:IsoAmyl alcohol-25:24:1) instead of DNA Salt Solution.
Neutralisation solution brings the pH back, resulting in precipitation of protein and genomic DNA
whereas PCI purifies nucleic acids and eliminates proteins and lipids.
P a g e | 28
CONCLUSIONBacterial DNA was isolated and stored. This bacterial DNA was further used in Gel
Electrophoresis and DNA Fingerprinting. (which has been explained later).
FORENSIC SIGNIFICANCEBacterial DNA is a novel avenue in forensic science. Bacterial DNA is more resistant to
environmental factors than human DNA and so can persist longer on a surface than human DNA.
The configuration of bacterial DNA is influenced by the surrounding environment and the
individuals microbiome. It is conceivable that the different bacterial patterns could discriminate
individuals with different lifestyles.[11]
SCOPE AND ADVANCEMENTBacterial DNA analysis will not replace standard DNA identification, but could become a
complimentary technique for when standard DNA identification provides only limited
information[11].A current is study being going to use bacterial fingerprint as human identification
tool in Forensic Science.
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PROCEDURE1. 1X TAE buffer was prepared from the given 50X TAE.
2. 0.7% of agarose was made in 1X TAE buffer. The volume of the buffer was depended on
the size of agarose gel prepared. 50 ml 1X TAE was taken and 0.35gm of agarose was
added to it.
3. The solution was heated in a microwave. It was continued until agarose had dissolved
completely.
4. Once the agarose had cooled to the point it could be held comfortably in hand, ethidium
bromide ( 2-4 micro liter) was added and agarose was poured into thegel casting mould,
using an appropriate size comb.
5. Once the gel set, the com was removed, transferred to the running apparatus and covered
with the running buffer until ready to use.
6. The amplified DNA samples (20 micro liter) were mixed with 15 micro liter of loading
dye and loaded in the wells.
RESULT AND DISCUSSIONElectrophoresis is used to separate charged molecules, whether they are DNA, RNA or proteins.
Agarose is a linear polymer which polymerizes into a semi-solid matrix on cooling. The resulting
matrix allows small molecules to move quickly through the gel, whereas the larger molecules are
hindered by the matrix, causing them to migrate slower. The addition of an electric current to the
gel causes the molecules to move in one direction, towards the positive end of the gel.
DNA molecules are negatively charged and therefore migrate through the Agarose gel matrix to
the positive terminal at the bottom of the gel.
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CONCLUSIONExtracted and isolated bacterial DNA was viewed under UV-Light and separation was observed.
(as shown in image).
FORENSIC SIGNIFICANCEElectrophoretic separation has uses in forensic science because it can be used to isolate and
compare DNA, blood proteins and inorganic substances such as gunshot residues from crime
scenes with suspects, victims or standard reference material. This process involves the separation
of 'marker' proteins that are found on the surface of red blood cells. Many of these are antigens
that determine particular blood groupings such as A, B, AB and O, and they can therefore be used
to exclude suspects from being present at a crime scene.[12]
RESULT AND DISCUSSIONFungal/ Plant DNA was extracted from the sample leaves given to us. Extracted Plant DNA can
be further used in Gel Electrophoresis as well as in DNA Fingerprinting. Sometimes plants
poisonous are used to commit a crime and they are also used as drugs of abuse, in such cases Plant
DNA Extraction can play a major role in investigation.
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FORENSIC SIGNIFICANCEOne study, published in the International Journal of Legal Medicine on February 29, shows the
potential of marijuana DNA to help solve related drug trafficking offenses, and another show how
pollen DNA can aid forensic investigations.
SCOPE AND ADVANCEMENTSA separate study by the same research team shows pine pollen is a reliable source of forensic DNA
evidence. Forensic experts value what small trace particles on clothing can tell them about a crime
scene. The recent Sam Houston State study found that pollen remained viable for DNA testing for
at least 14 days on clothing made of cottonmeaning pollen grains could potentially be used to
link a perpetrator or victim to a crime scene.[13]
DNA FINGERPRINTING
AIM- To perform DNA Fingerprinting on the given DNA sample.
PROCEDURE1.
2.
3.
4.
Labeled the tubes and added 20ul of DNA, 5ul EcoR1 and 10ul 4X buffer.
Mixed the contents of each tube by gently pipetting 4-5 times.
Placed each tube in a incubator at 37c for 24 hours.
The restricted DNA bands can be now visualized on a 1% agarose gel.
RESULT AND DISCUSSIONDNA Fingerprinting results in different sized fragments, these different sized fragments are called
restriction fragment length polymorphisms, or RFLPs. Everyone has genetic sequences called
P a g e | 32
variable number tandem repeats, or VNTRs Everyone has different amounts of VNTRs The
VNTRs make the different sized RFLPs.
FORENSIC SIGNIFICANCEThis process is used as one means of identification when an attacker or assailant has left some kind
of bodily fluid or blood at the scene of a crime and when no visual identification is possible. DNA
Fingerprinting is a very reliable and accurate technique.
P a g e | 33
When Anti-A was added to the first drop of blood, it showed agglutination whereas when Anti-B
was added to the second drop of blood it didnt show any agglutination in it. After that, when AntiD was added to the third drop it also showed agglutination. This means that the blood sample had
Antigen A in its red blood cells and Antibody- B in its plasma cells, thats why when we add
Anti-A to the blood drop it shows agglutination and also its a positive blood group because it
shows agglutination upon adding Anti-D antisera to it.
CONCLUSIONFrom the above observations, the blood sample had A positive blood group.
FORENSIC SIGNIFICANCE-
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Blood typing is a method to tell what specific type of blood you have. What type you have depends
on whether or not there are certain proteins, called antigens, on your red blood cells. Reverse
grouping or serum grouping is done from serum or plasma for the purpose of person identification.
Blood grouping has important role in the serological identification of disputed individual with
biological relationship i.e. disputed paternity, maternity etc. Grouping is done not only from whole
blood, red cell, plasma or serum, but also from body fluid such as seminal fluid, vaginal fluid,
urine, saliva and gastric juice. We can also demonstrate the grouping from body tissue nail, hair,
bone, dental tissue, soft tissue and also from stains. Blood and other body fluid collected from
victim and also from criminal has important role in the identification and investigation of criminal
cases. If we know the blood grouping of criminal beforehand and preserved the grouping sample
from all the listed criminal for grouping and DNA typing and also for future need then we can able
to identify the criminal after criminality as because criminal always put some evidence on the
victim about his offence during criminality. It will thus help our investigating department for the
detection, identification and investigation in the case of criminality.[11]
SCOPE AND ADVANCEMENTMurder, rape, kidnapping and hijacking are common normal phenomenon in this modern
civilization and in our day to day life. Modern science creates new weapons creating newer type
of offences but fail to reduce crimes and offences. We can reduce this by creating awareness among
general people about offences and crime, strengthening their unity against crimes. By introducing
blood group serology in the detection, identification and investigation of criminal cases. Or by
establishing forensic science laboratory in the country with 'collaboration between Transfusion
Medicine Department and Department of Medical jurisprudence and toxicology under home
ministry or with the help of other SARC countries or opening the department of criminology in
our country.[11]
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OD of test
Hb in gm% = ____________ * concentration of standard * dilution
OD of STD
Screen for, diagnose, and measure the severity of anemia (low RBCs, hemoglobin and
hematocrit) or polycythemia(high RBCs, hemoglobin and hematocrit)
Monitor the response to treatment of anemia or polycythemia
Help make decisions about blood transfusions or other treatments if the anemia is severe.
This test can indicate if there is a problem with red blood cell production and/or lifespan, but it
cannot determine the underlying cause. In addition to the full CBC, some other tests that may be
performed at the same time or as follow up to establish a cause include a blood smear, reticulocyte
count, iron studies, vitamin B12 and folate levels, and in more severe conditions, a bone
marrow examination.[12]
P a g e | 36
RBC COUNTAIM- To count Red Blood Cells in the given blood sample.
PROCEDURE1. Blood was drawn to the 0.5 mark in the RBC pipette.
2. After that diluting fluid (it is an isotonic solution that prevents haemolysis of blood, for eg.
Haymes fluid, it contains NaCl (0.5gm), Na2SO4 (2.5gm), Hg2Cl2 (0.25gm) in 100ml of
distilles water) was drawn to the 101 mark and it was shaken for 3 minutes.
3. The chamber was charged/ loaded with blood.
4. RBCs were counted using 40X in the 80 smallest squares.
P a g e | 37
FORENSIC SIGNIFICANCERBC count can help in determining a persons physical condition, such as whether the person was
anemic or not. Red blood cells, the most prevalent blood cells in the human body, are the primary
means of delivering oxygen from the lungs to the body tissues via the blood. For red blood cells,
the forensic analyst searches for smaller chemical substances residing on their surfaces, such as
antigens, which also tend to have important forensic implications.[13]
RESULT AND DISCUSSIONBlood contains an enzyme called catalase, which breaks down hydrogen peroxide into water and
oxygen gas.
2H2O2
catalase
H2O + O2
P a g e | 38
When this reaction occurs, the oxygen gas is released as bubbles. The catalase enzyme performs
an important function to living organisms because hydrogen peroxide is very toxic to living cells.
Other organisms, including plants and some bacteria, also make catalase. If you place a few drops
of hydrogen peroxide on a substance that contains catalase, it will bubble profusely. These
substances that bubble with the addition of hydrogen peroxide are said to test positive for catalase.
FORENSIC SIGNIFICANCEForensic scientists perform blood stain analysis to determine whether or not blood is present at
crime scene or not. Blood is the most well known and most significant evidence in the modern
criminal justice system. Blood evidence is important to the forensic investigator because it can link
a victim to a suspect. In forensic science blood has always been consider as class evidence. In fact
in some cases, forensic serologists were able to link single perpetrator to a blood stain with strong
probability estimates. During blood stain analysis, the forensic investigator find out is the sample
blood or its an animal blood. If its an animal blood then from what species did it come from and
if it is a human blood then they determine the age, sex, race of the source if the blood.[14]
SCOPE AND ADVANCEMENTSOnce you know that a stain is blood, there is a lot of potential information in a blood stain. A forensic
serologist can reveal:Pattern and shape: The shape and pattern of blood drops can reveal important information about the nature
of the wound from which the blood came.
Type: Blood typing can be used as an initial test to exclude some suspected sources of a bloodstain. For
example, if a blood stain at the crime scene contains Type A blood, but the key suspect has Type O blood,
the suspect could be excluded as a source of the blood stain meaning he or she definitely did not leave
P a g e | 39
the blood stain. Invisible blood stain patterns: invisible blood stains patterns can also be detected by using
the luminol test. Luminol, a chemical sprayed on carpets and furniture, reveals a slightly phosphorescent
in the dark where blood stains are present.[13]
DNA ISOLATION FROM BLOODAIM- To isolate DNA from the given blood sample.
PROCEDURE1. 0.3ml of blood sample was taken in a 2ml eppendorf tube and 0.9ml of RBC lysis buffer
was added, mixed by inverting the tube and then 50 micro liter Triton X was added and
mixed by inverting the tube several times.
2. Incubated for 5 minutes at room temperature to lyse the RBCs.
3. Centrifuged at 10,000 rpm for 5 minutes and the supernatant was discarded.
4. Again RBC lysis buffer and 30 micro liter of Triton X was added in the above pellet, mixed
and incubated for 5 minutes at room temperature.
5. Centrifuged at 10,000 rpm for 5 min and the supernatant was discarded ( RBC lysis was
completed and a white pellet of WBCs obtained).
6. In the cell pellet 0.3ml of WBC lysis buffer was added and mixed by inverting the tube and
then 40 micro liter of 10% SDS mixed properly by inverting the tubes several times.
7. Incubated for 5 minutes at room temperature.
8. At the end of the incubation 100 micro liter of NaCl was added and mixed properly and then
centrifuged at 10,000 rpm for 5 minutes.
9. The supernatant was transferred into a new tube and 300 micro liter of isopropanol was
added and mixed by inverting the eppendorf slowly and centrifuged at 10,000 rpm for 10
minutes.
10. The supernatant was discarded carefully and the tube was kept open at room temperature
until the pellet completely dried.
11. 20 micro liter TE buffer was added and stored at -20 degree C for storage.
RESULT AND DISCUSSIONBlood samples were taken to extract the DNA from them. DNA can be extracted only from the
WBCs cells and this DNA can also be used for individualization purposes by performing DNA
fingerprinting.
P a g e | 40
FORENSIC SIGNIFICANCEBlood contains DNA, and depending on the size of the stain and its condition (old, new, dry, etc.),
a forensic scientist may be able to get enough information to obtain a highly probable match of a
suspect with the evidence. Two techniques are heavily used by forensic scientists in evaluating
DNA evidence from blood or other body tissues polymerase chain reaction (PCR) and variable
number tandem repeats (VNTRs).[14]. Human DNA can be extracted from all the nucleated cells
such as hair, tissue, blood etc. certain sources contain high levels of proteins & many types of
secondary metabolites that effects DNA purification, highly purified DNA is essential for
molecular studies.
SCOPE AND ADVANCEMENTSIsolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic
research and analysis. DNA of hair samples can also be used for the genomic disorder analysis in
addition to the forensic analysis as a result of the ease of sample collection in a noninvasive
manner, lower sample volume requirements, and good storage capability. DNA typing is currently
the most validated method for the personal identification of human bodily fluid stains found at
crime scenes. DNA isolation using buccal swabs provides many advantages, such as cost-effective
processing, lower sample volume requirement, long-term archiving, and suitability of selfcollection. It is more comfortable for the patient, and the buccal swabs provide sufficient DNA for
the PCRs, as they demands only a few nanograms of DNA.[15]. Also, if we increase the database
systems, then storing and comparing the information of DNA samples would be more easier for
the forensic investigator to solve the crime.
P a g e | 41
FORENSIC ANALYSIS OF URINEAIM- To estimate the protein in urine sample through qualitative and quantitative methods.
QUALITATIVE METHOD ( Heating Method )PROCEDURE1. A test tube 2/3rd filled with urine was taken.
2. Upper portion of urine was boiled for 2 minutes (lower portion was not heated so that it
could be used as a control for comparing).
3. Appearance for turbidity was noted. (Turbidity can arise because of phosphates, carbonates
or proteins).
4. To confirm for presence of proteins a few drops of 10% acetic acid was added.
5. Turbidity was developed implies proteinuria.
RESULT AND DISCUSSIONThrough this method we can find out the amount of protein in urine. With this amount we can find
out was the death due to kidney failure or not. Proteins leaks into the urine if the kidneys are
damaged.
Proteins present in urine are Albumin or Keratinin.
P a g e | 42
RESULT AND DISCUSSIONThe biuret test is a chemical test used for detecting the presence of peptide bonds.in the presence
of peptides, a copper (II) ion forms a violet colored complex in an alkaline solution. The biuret
reaction can be used to assay the concentration of proteins because (for most proteins) peptide
bonds occur with approximately same frequency per gram of material. The intensity of the color,
and hence the absorption at 540nm, is directly proportional to the protein concentration, according
to the Beer- Lambert law.
When biuret reagent was added to the solution containing peptide bonds, the solution turned into
a violet color. The violet color was a positive test for the presence of protein. The more intense the
color, the greater the number of peptide bonds present.
P a g e | 43
S.NO
Protein (ml)
Conc.
Of Distilled
protein (mg) water
Biurette
reagent
O.D.
540nm
1.
0.0
0.0
2.0
3.0
0.0
2.
0.2
1.0
1.8
3.0
0.05
3.
0.4
2.0
1.6
3.0
0.09
4.
0.6
3.0
1.4
3.0
0.11
5.
0.8
4.0
1.2
3.0
0.19
CONCLUSIONThe optical densities of the sample was calculated and compared with the concentration of protein
present in the standard table.
FORENSIC SIGNIFICANCEUrinalysis can disclose evidence of diseases; even some that have not cause significant signs or
symptoms. Therefore, a urinalysis is commonly a part of routine health screening. A protein urine
test measures the amount of protein present in urine. Normally, healthy people dont have protein
in their urine. However, protein may be excreted in the urine when the kidneys arent working
properly or when high levels of certain proteins are present in the bloodstream.[16]. By collecting
and testing the urine samples, a forensic scientist can reveal that the victim or the assailant was
having any kidney infection or not. Then by collecting the urine samples from the suspects, he can
compare the results.
SCOPE AND ADVANCEMENTSBy performing protein test in the urine, a physician can determine
UTI
kidney infection
diabetes
dehydration
at
P a g e | 44
THIN LAYER CHROMATOGRAPHYAIM- To detect the presence of nicotine in the given urine sample by TLC.
PROCEDURE1. A clean glass slide was taken.
2. Took a beaker and 1.5gm of silica powder was weighed and added 4ml water, made a slurry
(thin paste).
3. The slurry was poured onto a glass slide and waited for dry at room temperature.
4. The glass pate was putted into a hot air oven at 55 degree C for 15-20 min for activation.
5. The solvent was prepared (Butanol : acetic acid : water in ratio 5 : 1 : 4).
6. A point was made on the plate with tip and loaded 10 15 micro liter sample.
7. The plate was then placed vertically into the solvent and covered it.
8. Waited for visualization of spots.
RESULT AND DISCUSSIONChromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated thin
layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer
with a thin layer of adsorbent material, usually silica gel, aluminum oxide or cellulose (blotter
paper). This layer of adsorbent is known as the stationary phase. After the sample has been applied
on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via
capillary action. Because different analytes ascend the TLC plate at different rates, separation is
achieved. To calculate the amount of nicotine in the urine Retention Factor was calculated.
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FORENSIC SIGNIFICANCETLC is a practical, economical, and sensitive method of detecting drugs in urine specimens. TLC
is particularly useful because multiple specimens can be tested, and more than one drug can be
determined for each application. The test involves applying urine specimens onto a glass plate,
which is sprayed with various reagents. There are TLC screening tests for most drugs of abuse,
including, the amphetamines, the barbiturates, cocaine, marijuana, glutethimide, and the
phenothiazines.
SCOPEThin layer chromatography can be used to monitor the progress of a reaction, identify compounds
present in a mixture, and determine the purity of a substance. Specific examples of these
applications include: analyzing the dye composition of fibers in forensics, assaying the
radiochemical purity radiopharmaceuticals or identification of medicinal plants and their
constituents.
P a g e | 46
RESULT AND DISCUSSIONThrough microscopic analyses we compared shape, dye content, size, chemical composition, and
microscopic appearances, yet all of this was still about "class evidence.
CONCLUSIONBy observing the above slide under 40X magnification in the microscope, the structure and scales
of fibers was detected which indicates that the given sample was Fiber.
FORENSIC SIGNIFICANCEHair and Fiber evidence at a crime scene can be used to link a suspect to a crime. Where the fibers are found
at a crime scene can also suggest the nature of contact that was made by the criminal. For example, fibers
found on a victim might suggest direct contact with that person, while fibers found in a victim's apartment
may only put the suspect in that location. Fibers are gathered at a crime scene with tweezers, tape, or
a vacuum. They generally come from clothing, drapery, wigs, carpeting, furniture, and
blankets. For analysis, they are first determined to be natural, manufactured, or a mix of both.
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SCOPE AND ADVANCEMENTSFiber evaluation can show such things as the type of fiber, its color, the possibility of violence,
location of suspects and the point of origin. This can also help in comparing and differentiating
natural fibers from the animal fibers as animal fibers contain protein in them. The problem with
fiber evidence is that fibers are not unique. Unlike fingerprints or DNA, they cannot pinpoint an
offender in any definitive manner. There must be other factors involved, such as evidence that the
fibers can corroborate or something unique to the fibers that set them apart.[17]. Apart from this,
if microscopic examinations would become more advanced and a proper data base would be
maintained, then the forensic scientist can use that data for comparision and they need not to collect
and re-compare all the samples again. They can also store this information for future use.
RESULTThe color of first eppendrof turned purple blue which indicated saliva is absent.
The color of second eppendrof turned yellow which indicated saliva is present.
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RESULTSmall blue color water like droplets was observed under 40X indicated saliva is present.
RESULTFruity smell was observed from the sample which indicated that the sample of saliva contained
alcohols.
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RESULTA precipitate was formed which indicated the presence of ethanol in the given saliva sample.
CONCLUSIONFrom the above 4 tests, it was concluded that the first 2 given samples was saliva and sample third
and fourth had the presence of alcohols in them.
FORENSIC SOGNIFICANCESaliva can be of forensic significance because traces of drugs that are circulating in the body can
be present in saliva. The composition of the saliva accurately mirrors the proteins that are present
in both the blood and urine. Thus, testing of saliva, which is easier and less obtrusive that obtaining
a blood or urine sample, can be used to reveal the presence of prescription and illicit drugs.
SCOPE AND ADVANCEMENTSThe test of saliva will enable the detection of viral and bacterial infections as well as diseases such
as cancer. These test are based on the presence of signature proteins that are unique to the maladies,
such as antibodies, from the microorganisms or the cancerous cells.
For example, an antibody-based saliva test for the human immunodeficiency virus (HIV; the
accepted cause of acquired immunodeficiency syndrome) is available for clinical use. No home
use tests are officially approved as of yet, although a number of no sanctioned and independently
evaluated tests are available through internet- based companies.
Promising preliminary research results published in February 2005 have shown that aberrant
genetic material (DNA) and the messenger ribonucleic acid (mRNA) that helps process the genetic
information into a protein from cancerous cells can also be detected in saliva. In the future, forensic
analysis of saliva may help determine if the subject has (or did have) cancer.
FORENSIC ANALYSIS OF DIATOMSAIM- To detect the presence of diatoms in the given tissue sample.
PROCEDURE1. A little piece of liver or kidney cut was taken and mashed into small pieces and putted in a
glass tube.
2. 1 ml of nitric acid solution was added to the tissue.
P a g e | 50
3.
4.
5.
6.
7.
The test tube was boiled using a test tube holder on a spirit lamp.
It was done carefully as nitric acid is a strong acid.
When the solution turned yellow, centrifuged the solution.
The supernatant was discarded and washed the pellet with distilled water.
The pellet was putted onto a glass slide and observed under microscope.
RESULT AND DISCUSSIONThe shells of diatoms was observed under 10X magnification under the microscope. The living
matter of each diatom is enclosed in a shell of silica that it secretes. These shells are marked by
minute pores or depressions that allow the living organism access to its environment. Diatoms
cells are contained within a unique silicate (silicic acid) cell wall. The characteristic feature of
diatoms is their ability to secrete an external wall composed of silica called Frustules.
FORENSIC SIGNIFICANCEDiatom test is based on a theory that normally people do not have significant number of diatoms
in some important organs like kidneys, liver, brain and bone marrow etc. but if a person drowns in
water containing diatoms, the diatoms in that water will reach the lungs and some of them will
penetrate the alveolar wall. If the heart is still beating, the diatoms that have entered the blood
stream will be transported around the body and may lodge in distant organs such as the kidneys,
liver, brain and bone marrow before death. Increase in the number of diatoms in the internal organs
was thought to confirm that the body had drowned and, if a sample of the water was also taken at
P a g e | 51
the presumed site of drowning, the similarity of different species of diatoms in the water and the
body could be determined. On the other hand, if a dead body was dropped into water, although
diatoms could reach the lung by passive percolation, no circulatory transfer could occur and so
(theoretically) no diatoms would be present in the distant organs. These are the only
microorganisms with acid resistant frustules, making them easier to extract from post-mortem
tissues. They are too small to penetrate various distant organs of the body. They can be easily
classified and identified.
SCOPE AND ADVANCEMENTSIt has been suggested that marrow of the sternum may be as good of a source of diatom as femoral
tissue. Data base can also be prepared of the location of diatoms found at crime scene for making
the future searches easier.
RESULT-
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By mixing the acid solution in the solution, the required pH of 7 was reached and the buffer was
prepared to store or to use.
IMPORTANCEBuffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of
chemical and biological applications. An example of a buffer solution is blood. Phosphate-buffered
saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a waterbased salt solution containing sodium hydrogen phosphate, sodium chloride and, in some
formulations, potassium chloride and potassium dihydrogen phosphate. The osmolarity and ion
concentrations of the solutions match those of the human body (isotonic). PBS has many uses
because it is isotonic and non-toxic to most cells. These uses include substance dilution and cell
container rinsing. PBS with EDTA is also used to disengage attached and clumped cells.[18]
PAPER CHROMATOGRAPHYAIM- To detect different ink in the given sample by paper chromatography.
PROCEDURE1. Thin strips of Whatmann filter paper was taken.
2. A spot at 1 cm was made above the paper edge using the pen/marker/sketch pen which was
found as evidence at the crime scene.
3. These paper strips were hanged in a beaker in a vertical position containing the mobile phase
facing the sample spots downwards.
4. The solvents used were Distill water
Butanol : acetic acid : water in the ratio 2:1:8
5. The beaker was covered with aluminium foil and waited until the solution ran till 3/4th length
of the slide.
6. Then these paper strips was taken out and dried them for 2 minutes.
7. The different spots were compared and observed in different paper trips and solvents.
RESULT AND DISCUSSIONRf (Retention Factor) = Distance travelled by band Distance travelled by Solvent
Rf value of sample 1 = 0.74
Rf value of standard O = 0.94
Rf value of standard B = 0.78
In the second sample Rf value of first ink was 0.36 and the Rf value of second ink was 0.91
P a g e | 53
CONCLUSIONSample matches the standard B (Rf =0.78), hence ink sample belonged to sample B.
FORENSIC SIGNIFICANCEPaper chromatography is an analytical method technique for separating and identifying mixtures
that are or can be colored, especially pigments. This technique has important use in forensic
sciences. Paper chromatography can separate minute amounts of substances. This makes it very
useful for forensics investigators. Drugs from narcotics to aspirin can be identified in urine and
blood by using chromatography. Detectives can analyse a sample of paint to find out the car it
came from, including the model and year it was made. Ink can be separated into its components to
help identify the pen used for ransom notes and forgeries, as well as the type of ink used for
counterfeiting.
SCOPE AND ADVANCEMENTSInk analysis is performed to determine if two inks are similar or different, to determine the
manufacturing source of an ink, and to date an ink. For the latter two tasks, it is necessary to have
a collection of reference standards; it is also needed when inks are found to be similar and one
needs to know how rare or common that ink is. The dating of inks is the most challenging and is
done via two approaches. The first uses a collection of reference standards. Here a match suggests
the identity of an ink for which one has the manufacturing information such as the first date of
production. The second involves the examination of features that change with age such as the
evaporation of ink solvents and the degradation of dyes. Within this approach, there are two subapproaches. One estimates the age of an ink by comparing the ink with itself before and after
inducing aging (e.g., by heating). The other determines the relative age of inks (some of which are
of known age) that are of the same formula and appear on the same paper.[19]
P a g e | 54
Paper chromatography can also be used for drug analysis, paint analysis,etc.
Sample (1ul) was loaded in sample injection port using a micro syringe.
Software for GC was run as soon as the sample was loaded to get precise result.
Sample was then compared with the standards.
P a g e | 55
P a g e | 56
FORENSIC SIGNIFICANCEGC is widely used by forensic scientists from analysis of body fluids for the presence of illegal
substances, to testing of fiber and blood from a crime scene, and to detect residue from explosives.
P a g e | 57
REFERENCES
[1] Codon Biotech Pvt Limited
[2] Anna Tectsov . Ink Analysis : The Vinland Map / The Mc crone group
[3] The Forensic Outbreak Team . 5 real life cases where DNA profiling charged everything
[4] HA Dealman . Fiber evidence and the Wayne Wiliams trid . 1984
[5] Abigail A. Salyers . Microbes in Court : The emerging field of Microbial Forensics . January
2004.
[6] Steven E. Schutzer, Rozer G. Breeze, Bruce Budowle . Microbial Forensics Program . 2005 .
page-8
[7] Maryann Depietro . Gram Stain . Part-1 , Part-2
[8] National Public Health Laboratory . Example Analysis SOP.pdf . August 1, 2013 . Page 1
[9] Nunh-huh . Talk- Gram Staining . July 2006 (UTC)
[10] Dr. Ciira Kiiyukia . Food Microbiology for Enthiopian Health and Nutrition Reasearch
Institute. December 2003 . Page 5
[11] Mondal AG, Islam MA, Rahman MM, Begum D, Sultan MT . Role of Blood Group Serology
in the Detection, Identification & Investigation for Criminality . July 4, 2011 . page 83,87
[12] Lab tests Online . Hemoglobin : The Test/ Hemoglobin Test : Hgb ; Hb; H and H/Lab .
October 29, 2015
[13] Auce Yang, Period 7 . Forensic Serology ; Blood
[14] Hughes ,Undergraduate Biological Science Education Initiative . Blood Stain Analysis (part
one)
[15] Souvik Ghatal, Rajendra Bose, Muthukumaran, Senthil, Kumar Nchimuthu . A simple method
of Genomic DNA Extraction from human samples for PCR RFLP Analysis . December,2013 .
Page 1
[16] Janelle Martel . Protein Urine Test . November 30, 2015 . Page 1,2
[17] Douglas W. Deedrick . Hairs, Fibers, Crime and Evidence Part:2 Fiber Evidence . July 2000volume 2 number 3 . Page 2
[18] Phosphate buffered Saline Wikipedia
[19] Antorio A. Cantu . Ink Analysis in : Encyclopedia of Forensic Science.