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ADVANCE TECHNIQUES IN FORENAIC SCIENCE

Submitted in partial fulfillment of requirements for the degree of B.Sc.
Honours in Forensic Science

Submitted by

Shourya Sharma
(Batch: 2014 - 2017)

Under the Supervision of

Dr. Mohit Soni

Department of Applied Chemistry, Amity School of Applied Sciences,

Amity University Haryana, July 2016

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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.

Date:1 July, 2016

Shourya Sharma

Place: Manesar, Gurgaon

A51605914008
Department of Applied Chemistry
Amity University Gurgaon
Haryana

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Department of Applied Chemistry

Amity School of Applied Sciences
CERTIFICATE
This is to certify that the work contained in the dissertation entitled Advance Techniques in
Forensic Science, submitted in partial fulfillment of the requirements for the award of the degree
of Bachelor of Science in Forensic Science is an authentic record of work carried out by Shourya
Sharma under my/our supervision. The matter embodied in this dissertation has not been submitted
elsewhere for a degree.

Mr.Ravi Rathi
Teaching Associate
Co-supervisor

Dr. Mohit Soni

Assistant Professor
Supervisor
Department of Applied
Chemistry

Prof. (Dr.) Anek Pal Gupta

Head
Department of Applied Chemistry
& Polymer Technology Centre

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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.

Amity University Gurgaon, Manesar

Date: 1 July, 2016

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

Characterization of Bacteria/Gram staining

21

Bacterial identification by IMViC

23

Bacterial Genomic DNA Isolation

27

Agarose Gel Electrophoresis

28

Plant Genomic DNA Isolation

30

DNA Fingerprinting

31

Blood Grouping

32

Haemoglobin Estimation

34

RBC Counting

36

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Blood Stain analysis

37

DNA estimation from Blood

39

Forensic analysis from Urine

41

Thin Layer Chromatography

44

Hair and Fiber analysis

46

Forensic analysis of saliva

47

Forensic analysis of Diatom

49

Preparation of phosphate buffer

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]

2. ISOLATION AND CHARACTERIZATION OF BACTERIA/GRAM

STAINING.
Gram staining is also called Grams Method; it is a method of differentiating bacterial species into
two large groups. The name comes from Danish bacteriologist Hans Christian Gram, who
developed the technique. Gram staining differentiates bacteria by physical and chemical properties
of their cell walls by detecting peptidoglycan, which is present in a thick layer in gram positive
bacteria.
GRAM POSITIVE MICROORGANISMS

GRAM NEGATIVE MICROORGANISMS

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.

3. Identification of bacteria by IMViC test:

The IMViC tests are a group of individual tests used in microbiology lab testing to identify an
organism in the coliform group. A coliform is a gram negative, aerobic or facultative anaerobic
rod which produces gas from lactose within 48 hours. The presence of some coliforms indicates
fecal contamination.IMViC is an acronym that stands for indole, methyl red, Voges-Proskauer and
citrate. To obtain the results of these four tests, three test tubes are inoculated: tryptone broth
(indole test), methyl red-Voges Proskauer broth (MR-VP) and citrate.
4. BACTERIAL GENOMIC DNA:
Bacterial cells are very small, roughly the size of an animal Mitochondrion (about 1-2um in
diameter and 10um long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and
spiral. Bacterial cell consist of a capsule, cell wall, and a cell membrane, a cytoplasmic region that
contains the cell genome, ribosome, and various sorts of inclusions appendages, sometimes
present, called Flagella and pili. Bacteria cell lack membrane bound nucleus but instead resides
inside the bacterial cytoplasm. Along with chromosomal DNA, most bacteria also contain small
independent pieces of DNA called PLASMIDS that often encode for traits that are advantageous
but not essential to their bacterial host. Plasmid can easily be gained or lost by a bacterium and
can be transferred between bacteria as a form of horizontal gene transfer. So plasmid can be
described as an extra chromosomal DNA in a bacterial cell.
5. AGAROSE GEL ELECTROPHORESIS:
Electrophoresis 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

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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.

8. FORENSIC ANALYSIS OF BLOOD:

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.
Blood is often grouped according to the ABO blood typing system. This method breaks blood
types down into four categories:
1. Type A
2. Type B
3. Type AB
4. Type O

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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.

10. RBC COUNT:

Red blood cells (also referred to as erythrocytes) are the most common type of blood cell and the
vertebrate organisms principle means of delivering oxygen (O2) to the body tissues via the blood
flow through the circulatory system. They take up oxygen in the lungs or gills and release it while
squeezing through the bodys capillaries. These cells cytoplasm is rich in haemoglobin, an ironcontaining biomolecules that can bind oxygen and is responsible for the bloods red color. In
human, mature red cells are flexible biconcave disks that lack a cell nucleus and most organelles.
The cells develop in the bone marrow and circulate for about 100-120 days in the body before their
components are recycled by macrophages. Each circulation takes about 20 seconds.
Approximately a quarter of the cells in the human body are red blood cells. Red blood cells are
also known as RBCs, red blood corpuscles, haematoid, erythroid cell or erythrocytes. A RBC count
is a blood test that tells how many red blood cells (RBCs) you have. This test can help diagnose
anaemia and other conditions affecting red blood cells. The RBC count is almost always part of
the CBC (complete blood count) test.

11. BLOOD STAIN ANALYSIS:

Investigators often find blood stains during their examination of a crime scene. They also find
stains that could be either blood or some other similar substance, like reddish-brown paint. Blood
contains an enzyme called catalase, which breaks down hydrogen peroxide into water and oxygen
gas.
2H2O2

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.

P a g e | 12

12. DNA ISOLATION FROM BLOOD:

Isolating DNA from both fresh and particularly from frozen, whole blood samples is very difficult.
Normal human blood consists of 45% erythrocytes and 54% thrombocytes, which contain no
DNA. The DNA-containing leukocytes consist of only 12% of all blood cells at 6.6 pg of DNA
per cell (5 x 106 to 107 cells/mL). In addition, blood typically contains 150 mg/mL of hemoglobin
and 6080 mg/mL of plasma protein. Human DNA can be extracted from all the nucleated cells
such as hair, tissue, blood etc. certain sources contain high levels of proteins and many types of
secondary metabolites that effects DNA purification, highly purified DNA is essential for
Molecular studies. Here followed salting out method to extract large quantities of human DNA
from whole blood, it is less time consuming and cost effective as well as gives better concentration
of DNA.

13. FORENSIC ANALYSIS OF URINE:

Urinalysis 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. Urinalysis is
also a very useful test that may be ordered by your physician for particular reasons. Urinalysis is
commonly used to diagnose a urinary tract or kidney infection, to evaluate causes of kidney failure,
to screen for progression of some chronic conditions such as diabetes mellitus and high blood
pressure (hypertension).

14. Detection of Drugs:

TLC:

Rapid method of Nicotine detection in Urine by

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.

16. FORENSIC ANALYSIS OF SALIVA:

Saliva is a clear liquid that is made and is present in the mouth, where it has a number of functions.
It wets food and makes the food easier to swallow. As well, specialized proteins that are present
in saliva trigger chemical reactions that begin to break apart chemical bonds in the food.
Saliva 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.

17. FORENSIC FIBER ANALYSIS:

Placing a suspect at the scene of a crime is an important element in criminal investigation. This
can be achieved through the location of textile fibers similar to those from the victim's clothing or
the crime scene on the clothing of the suspect, or through the discovery of fibers like those in the
suspect's clothing at the crime scene. When fibers are matched with a specific source (fabric from
the victim, suspect, and/or scene), a value is placed on that association. Whether a fiber is
transferred and detected is dependent on the nature and duration of contact between the suspect
and the victim or crime scene, the persistence of fibers after the transfer, and the type(s) of fabric
involved in contact. Fibers are considered a form of trace evidence that can be transferred from the
clothing of a suspect to the clothing of a victim during the commission of a crime. Fibers can also
transfer from a fabric source such as a carpet, bed, or furniture at a crime scene. These transfers
can either be direct (primary) or indirect (secondary). A primary transfer occurs when a fiber is
transferred from a fabric directly onto a victim's clothing, whereas a secondary transfer occurs
when already transferred fibers on the clothing of a suspect transfer to the clothing of a victim. An
understanding of the mechanics of primary and secondary transfer is important when
reconstructing the events of a crime.
18. FORENSIC ANALYSIS OF DIATOMS:
Diatoms are unicellular organisms of the kingdom Protista, characterized by a silica shell of often
intricate and beautiful sculpturing. Most diatoms exist singly, although some join to form colonies.
They are usually yellowish or brownish, and are found in fresh and saltwater (Plate-1), in moist
soil, and on the moist surface of plants. 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.
While diatom communities exist almost everywhere, the communities that live in water under
natural conditions usually consist of more species than those communities found in aerial habitats

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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.

19. PREPARATION OF PHOSPHATE BUFFER:

A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate
base or a weak base and its conjugate acid. It has the property that the pH of the solution changes
very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a
means of keeping pH at a nearly constant value in a wide variety of chemical and biological
applications.
The most commonly used phosphate buffers, consist of a mixture of monobasic dihydrogen
phosphate and dibasic monohydrogen phosphate. By varying the amount of each salt, a range of
buffers can be prepared that buffer well between pH 5.8 and pH 8.0. The buffer is most commonly
prepared at pH 7 using monosodium phosphate and its conjugate base, disodium phosphate.

20. ANALYSIS OF ALCOHOL BY GAS CHROMATOGRAPHY:

Gas chromatography specifically gas- liquid chromatography involves a sample being
vaporised and injected onto the head of the chromatographic column. The sample is transported
through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid
stationary phase which is adsorbed onto the surface of an inert solid.

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.

Sample injection port

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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 open tubular (WCOT)

Support coated open tubular (SCOT).

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

P a g e | 16

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:

Flame ionization (FID)

Thermal conductivity(TCD)
Electron capture (ECD)
Flame photometric(FPD)
Photo-ionization(PID)
Hall electrolytic conductivity
Nitrogen - phosphorus

Flame ionisation detector

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]

2. DNA Fingerprinting evidence in the Richard Buckland case:

Fifteen year old school girl, Lynda Mann was abducted in Narbourough, England. The next day,
her body was discovered raped and murdered. Three years later, another young woman met the
same fate near Lyndas resting place. Richard Buckland was arrested and confessed to the second
murder only. An untested technique was applied; genetic fingerprinting through DNA analysis.
Surprisingly, there was no match in either murder, so the test was repeated. Ultimately, Buckland
was proven innocent. As to why he confessed, he claimed he had been pressured by police. 5,500
men from the local area were then tested. Colin Pitchfork persuaded a friend to test in his place,
but when he bragged about fooling the investigators, he was overheard and reported. His genetic
profile matched the semen samples from both girls, and in 1987 he became the first murderer
convicted by DNA.[3]

3. Ink analysis in the case of Vinland Map:

The Vinland Map was found inside a book entitled The Tarter Relation, both of which were
donated to Yale University in the late 1950s. In 1965, after seven years of analysis, Yale University
Library revealed the Vinland Map to the world and claimed that it was genuine. The Yale analysis
suggested the Viking voyage preceded Columbus by at least 50 years. Shortly after Yales claim

P a g e | 18

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.

Composition of nutrient agar : NaCl-8gm

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

P a g e | 20

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.

Fig 1:- Nutrient Broth was prepared

Fig 2:- Nutrient Agar was prepared

CONCLUSIONNutrient broth and nutrient agar media were prepared.

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]

ADVANCEMENTThe burgeoning field of microbial forensics should be accompanied by a parallel development of

educational infrastructure and resources targated at the next generation of practitioners as well as
diverse elements of the policy, research and the law enforcement communities. A microbial
forensics program can be broad, providing information encompassing all aspects of the field from
science to policy, or more focused depending on its purpose and target audience. This student may
become a forensic scientist analyzing crime scene evidence for a law enforcement and intelligence
agency whose responsibility is to understand the scope of an investigation and what tools are
available to generate investigative leads.[6]

ISOLATION AND CHARACTERIZATION OF

BACTERIA / GRAM STAINING
AIM- To isolate and characterize the given bacterial culture.
PROCEDURE1.
2.
3.
4.
5.
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.

P a g e | 22

8. Countered stained with saffranin for 45 sec.

9. The slide was then air dried and mounted.

RESULT AND DISCUSSIONGram staining - Gram staining was done & the stained bacterial smears were visualized under
microscope and differentiate gram positive and gram negative bacteria.

Fig 3:- Gram negative bacteria

Fig 4:- Gram positive 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

P a g e | 23

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]

IDENTIFICATION OF BACTERIA BY IMViC TEST

AIM- To identify the given bacterial culture by IMViC Tests.
THE INDOLE TESTTryptone broth:

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.

The Methyl-Red and Voges-Proskaeur Tests:

MRVP Broth:
Peptone
Glucose/dextrose
Potassium Phosphate
Distilled water

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.

P a g e | 24

3. Inoculated with required culture and incubated at 370 C for 48 hours.

FOR MR TEST1. After 48 hours 5 drops of methyl red solution were added to the tube and the formation
of red color of the broth was observed. This showed that MR test is positive.
FOR VP TEST1. After 48 hours 12 drops of VP reagent I solution were added and then 2 3 drops of
VP reagent II (Together known as Barittes reagent) were added to the tube and the
formation of pink to red color of the broth was observed. This showed that the VP
test is positive.
VP reagent 1- 6gm alpha napthol in 100ml 95% alcohol.
VP reagent II 16gm KOH in 100 ml distilled water.

CITRATE TESTCITRATE AGARAmmonium dihydrogen

phosphate
Dipotassium phosphate
Sodium citrate
Sodium Chloride
Magnesium Sulphate
Agar
Bromothymol blue
Distilled water

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.

RESULT AND DISCUSSION-

P a g e | 25

BACTERIAS
Ecoli

Enterobacter

Bacillus

Klebsiella

MR

VP

v/+/-

TEST
INDOLE

CITRATE

TABLE 1:- IMViC Tests Results

The Indole Test:

The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan.
Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves
tryptophan, producing indole and other products. When Kovacs reagent (pdimethylaminobenzaldehyde) is added to a broth with indole in it, a dark red (cherry) colour ring
develops.

The Methyl-Red and Voges-Proskaeur Tests:

MR-VP media contains glucose and peptone. All enterics oxidize glucose for energy; however the
end products vary depending on bacterial enzymes. Both the MR and VP tests are used to
determine what end products result when the test organism degrades glucose. E.coli is one of the
bacteria that produces acids, causing the pH to drop below 4.4.When the pH indicator methyl red
is added to acidic broth it will be cherry red (a positive MR test).
Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl alcohol,acetyl
methyl carbinol). In the neutral pH the growth of the bacteria is not inhibited. The bacteria thus
begin to attack the peptone in the broth, causing the pH to rise above 6.2.At this pH, methyl red
indicator is a yellow colour( a negative MR test).
The reagents used for the VP test are Barritts A (alpha-napthol) and Barritts B (potassium
hydroxide).When these agents are added to a broth in which acetyl methyl carbinol is present they
turn a pink-burgundy colour (a positive VP test).

The Citrate test:

The citrate test utilizes Simmons citrate media to determine if a bacterium can grow utilizing
citrate as its sole carbon and energy source.Simmons media contains bromothymol blue, a pH
indicatorwith a range to 6.0 to 7.6. Bromothymol blue is yellow at acidic pH (around 6) and
gradually changes to blue at more alkaline pH ( around 7.6). Uninoculated Simmons 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.

SCOPEThe scope of IMViC tests are as follows

To meet certain set standards

To estimate the shelf-life of the product

P a g e | 27

To determine quality of the food

For public health purposes
To identify the food poisoning orgainsms
To determine the infectious organisms
To identify the spoilage organisms[10].

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

Fig 6:- Isolated Bacterial Genomic DNA

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.

AGAROSE GEL ELECTROPHORESISAIM- To prepare Agarose Gel Electrophoresis.

P a g e | 29

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.

Fig 7:- DNA bands under UV light

P a g e | 30

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]

EXTRACTION OF PLANT GENOMIC DNA

AIM- To extract Plant Genomic DNA
PROCEDURE1. 100mg of fungal culture/plant tissue was taken in mortar and macerated/grinded it with the
help of a pestle and 500l of CTAB buffer was added to the tube.
2. Inverted the tube several times to slowly mix and kept at 65c for 30 min.
3. Added 0.2ml DNA salt solution to the tube and mixed it by inverting several times.
4. Centrifuged the tube at 10000 rpm for 10 min and collected the supernatant carefully.
5. Added 0.8 ml DNA precipitating solution then slowly inverted the tube several times to
mix, observed the appearance of white thread like strand.
6. The DNA was centrifuged at high speed for at least 10 min.
7. Removed the supernatant and washed the pallet with 0.5ml of 70% ethanol and centrifuged
at high speed for 10min.
8. Discarded the ethanol and kept the tube open at room temp until it was dry.

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.

P a g e | 31

Fig 8:- Extracted Plant Genomic DNA

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.

Fig 9:- Fragments of DNA under UV light

CONCLUSIONDNA sequence/fragments were viewed under UV-Light.

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.

FORENSIC ANALYSIS OF BLOOD

AIM- To detect the blood group and Rh factor from the given blood sample.
PROCEDURE1.
2.
3.
4.
5.

Oxalated whole blood was collected.

Marked one slide to the left with Anti-A and the right side with Anti-B.
Another slide was marked with Anti-D.
A drop of respective antisera was added to each of the marked position on the slide.
Carefully without disturbing the slide a drop of blood was added to each of the anti-sera on
the slide.
6. Mixed well with separate applicator sticks and observed for agglutination for 2 minutes.

P a g e | 33

RESULT AND DISCUSSION-

Fig 10:- Table of blood grouping

Fig 11:- Blood Grouping

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-

P a g e | 34

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]

HAEMOGLOBIN ESTIMATIONAIM- To estimate Haemoglobin amount in the given blood sample.

PROCEDURE1.
2.
3.
4.
5.
6.
7.

Ring finger was cleaned with 70% alcohol.

Pricked finger with a needle and discarded the first drop of blood.
20ul of blood was withdrawn using an Hb pipette.
The drawn blood was mixed with 5ml of Drabkins reagent and mixed this gently in a tube.
Incubated for 10 minutes at room temperature.
The OD of the solution was taken at 540nm using green filter in colorimeter.
The amount of hemoglobin was then calculated by using the following equation:

P a g e | 35

OD of test
Hb in gm% = ____________ * concentration of standard * dilution
OD of STD

RESULT AND DISCUSSIONOD of test = 0.31

OD of STD = 0.35
Concentration of standard = 60
Dilution factor = 250/1000 (Dilution factor = Blood + Drabkins reagent/ 1000)
By putting all these calculations in the above formula , we gotHb in gm% = 0.35 0.31 * 60 * 250 1000

CONCLUSIONPercentage of hemoglobin in blood sample came out to be 13.28%.

FORENSIC SIGNIFICANCEThe haemoglobin test is often used to check for anemia, usually along with a hematocrit or as
part of a complete blood count (CBC). The test may be used to screen for, diagnose, or monitor
a number of conditions and diseases that affect red blood cells (RBCs) and/or the amount of
hemoglobin in blood. Hemoglobin is the iron-containing protein found in all red blood cells that
enables RBCs to bind to oxygen in the lungs and carry it to tissues and organs throughout the body.
By estimating the percentage of hemoglobin in blood, a forensic scientist can determine that the
victim or the suspect was anemic or not. This can also help them to identify the type of disease
related to human blood and percentage of hemoglobin.[12]

SCOPE AND ADVANCEMENTSA hemoglobin test may be used to:

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.

Fig 12:-Diagram of chamber

CalculationsRBC Count=No. of cells counted X Dilution Factor (Dilution Factor= Diluting fluid = 100
Area Counted X Depth Factor
Blood
0.5
Depth was 1/10mm and Area counted = 80/400 = 1/5 sq mm.
So, the final formula came out to be:
RBC Count=No. of cells counted X 200 = No. counted X 10,000
1/5 X 1/10

RESULTResults may vary, but in general:

Female: 4.2 to 5.4 million cells/ml
Male: 4.7 to 6.1 million cells/ml

P a g e | 37

Fig 13:-RBC in one of the blocks of chamber

CONCLUSIONRBC count in the given blood sample was came out to be 4.8 million.

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]

BLOOD STAIN ANALYSISAIM- To identify the blood stains.

PROCEDURE1. Each of the stains were tested to see if it was catalase positive or negative.
2. Few drops of hydrogen peroxide was placed on the small amount on each stain.
3. Results were recorded in a table.

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.

Fig 14:- Result of Blood Stain Analysis

CONCLUSIONBlood stain was identified by using H2O2.

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

Fig 15:- DNA extracted from blood

CONCLUSIONDNA was extracted from the given blood sample.

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.

Fig 16:- Test of Protein

CONCLUSIONTurbidity occurred in the urine sample because of presence of carbonates, phosphates or protein
which implied proteinuria. Proteinuria is often a sign of kidney damage and disease.

P a g e | 42

QUANTITATIVE METHOD- ( BIURET TEST )PROCEDURE1. 5 test tubes were taken.

2. Then, biuret reagent was prepared by adding copper sulphate, sodium potassium tartarate
and potassium hydroxide.
3. Stock of egg albumin was prepared by adding 0.05gm of egg albumin in 10 ml of distilled
water.
4. Now, in the first tube, 2ml of distilled water and 3ml of biuret reagent was taken and covered
with foil paper.
5. In second test tube, 0.1ml of protein sample (egg albumin) was taken and 1.8 ml of distilled
water and 3ml of biuret reagent was added and covered with the foil paper.
6. In third test tube, 0.4 ml of protein sample was taken and 1.6ml of distilled water and 3ml
of biuret reagent was added and covered with foil paper.
7. In fourth test tube, 0.6ml of protein sample was taken and 1.4 ml of distilled water and 3ml
of biuret reagent was added and covered with the foil paper.
8. In fifth test tube, o.8ml of protein sample was taken and 1.2ml of distilled water and 3ml of
biuret reagent was added and covered with foil paper.
9. Then these all five tubes were kept in incubator at 37 degree C for 10 minutes.
10. Took the first test tube (blank) and set the colorimeter at zero at 540nm.
11. After that, optical density of rest of the test tube at 540nm was recorded.

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.

Fig 17:- Colorimeter

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

amyloidosis (a build-up of protein in the bodys tissues)

drugs that damage the kidneys (such as NSAIDs, antimicrobials, diuretics, and chemotherapy
drugs)
hypertension (high blood pressure)
preeclampsia (high blood pressure in pregnant women)
heavy metal poisoning
polycystic kidney disease
congestive heart failure
glomerulonephritis (a kidney disease that causes kidney damage)
systemic lupus erythematosus (an autoimmune disease)
Goodpasture syndrome (an autoimmune disease)
multiple myeloma (a type of cancer affecting bone marrow)
bladder tumor or cancer[16]

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.

Rf = Distance moved by the molecule (location of the spot)/

Distance moved by the mobile phase (solvent front)

P a g e | 45

Fig 18:- TLC Results

CONCLUSIONThe Rf value of first sample came out to ne 0.52 and the Rf value of second sample came out to
be 0.46.

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

HAIR AND FIBER ANALYSIS

AIM- To view the fiber evidence under the microscope.
PROCEDURE1. A clean glass slide was taken.
2. 2-3 drops of colorless nail polish was putted on it and spread evenly.
3. The fiber/hair was placed on the slide for 10 minutes and allowed it to dry at room
temperature.
4. The fiber/hair removed carefully and the scale impressions was observed under the
microscope under 10X magnification.

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.

Fig 19:- Fiber slide

Fig 20:- Structure of Fiber under 40x

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.

P a g e | 47

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.

FORENSIC ANALYSIS OF SALIVAAIM- To detect the presence of Saliva.

SALIVA TEST 1- ( to check the presence of saliva)
PROCEDURE1. 2 eppendrorfs were taken and 1 ml of saliva was added in one tube and 1 ml of distilled
water in the other.
2. To both the samples 10 drops of 1% starch solution was added.
3. Next 2 drops of iodine solution was added.

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.

P a g e | 48

Fig 21:- Presence of saliva

SALIVA TEST 2- ( to check the presence of saliva)
PROCEDURE1. A sublingual saliva sample (saliva secreted by glands located below the tongue) was
collected.
2. A dab of saliva was collected and placed it on the surface of the slide.
3. Allowed the sample to dry for at least 5 min before attempting to read results.
4. The pattern formed by the saliva under 40X was observed. (fern like appearance).
5. Next a drop of methylene blue was added to the slide for 1 min.
6. The dye was allowed to drain off and added 1 ml of distilled water to was excess stain.
7. Observed at 40X.

RESULTSmall blue color water like droplets was observed under 40X indicated saliva is present.

SALIVA TEST 3- ( to check the presence of alcohols in saliva)

PROCEDURE1.
2.
3.
4.

1 ml of sample was taken in a glass tube.

To the given sample one drop of acetic acid and one drop of sulfuric acid was added.
The tube was heated gently and then cooled down.
The tube was gently swirled and noted for the presence of fruity smell.

RESULTFruity smell was observed from the sample which indicated that the sample of saliva contained
alcohols.

SALIVA TEST 4- (to detect the presence of ethanol)

PROCEDURE-

P a g e | 49

1. To an empty tube, 1 ml of 5% NaOH and 1-2 iodine crystals was added.

2. The tube was swirled to completely dissolve the iodine crystals.
3. The 2ml sample was added to the sides of the tube and noted for the formation of a
precipitate.

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.

Fig 22 :- Shells of Diatoms under 40X magnification

CONCLUSIONBy observing the different shells of diatoms under the microscope it was concluded that the given
sample of tissue contained diatoms in it.

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.

PREPARATION OF PHOSPHATE BUFFERAIM- To prepare phosphate buffer.

PROCEDURE1. 50 ml of 0.1M solutions of both acetic acid and base salts were made and then started
adding the acid to the base solution.
2. The pH meter was adjusted with standard buffer and then adjusted the test solution.
3. The two solutions was mixed continuously and monitored the pH change.
4. When the required pH of 7 reached, mixing the acid solution was stopped and the buffer
was to be used or stored.

RESULT-

Fig 23 :- The pH Meter

CONCLUSION-

P a g e | 52

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

Fig 24 :- Paper Chromatography of Ink

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.

ANALYSIS OF ALCOHOL BY GAS CHROMATOGRAPHY

AIM- To measure the amount of Methanol in the given alcohol sample.
PROCEDRURE

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.

RESULTStandards of Ethanol, Acetone and Methanol

P a g e | 55

P a g e | 56

Concentration of Sample= Molecular Weight of Sample X Area of Sample

Area of Standard
Concentration of Ethanol= 46 X 8991277 = 8.1 gm/l
51034247
Concentration of Methanol= 32.04 X 142407 = 0.09 gm/l
48205566

CONCLUSIONThe given alcohol contains methanol of concentration 0.09 gm/l.

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.