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EXAMINATION OF SALIVA STAINS

INTRODUCTION

Identity means the determination of the individuality of a person. The question of

identification of a living person is mostly the concern of the police and is raised in
criminal courts in connection with absconding soldiers and criminals, or persons
accused of assault, rape or murder; or when there is a mix-up of new born babies in
hospitals, or young lost children; and occasionally, in adults who have lost their
memory. It is also frequently used in civil courts owing to impersonation practised by
people to secure unlawful possession of property, insurance claims or to obtain the
prolongation of a lapsed pension.

One of the methods of personal identification is by examination of saliva stains.

WHAT IS SALIVA?

1) watery, frothy substance

2) produced in salivary glands
3) It is 98% water.
4) contains important substances:
mucus,
antibacterial compounds and
various enzymes
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Saliva is a thin fluid secreted by salivary glands. It contains enzymes like ptyalin,
glucose-6-phosphate dehydrogenase, various proteins, lipids, chloride and
thiocyanate ions.

BIOLOGICAL CHARACTERISTICS

1) Humans produce 1 -1.5 L of saliva per day.

2) 70% is produced in submandibular gland.
3) 25% is produced in parotid gland.
4) 5% is produced in sublingual gland.
5) It aids in digestion by lubricating food and begins breakdown of starches.

SALIVA STAINS:

Saliva stains may be found:

1) At Scene.
2) On handkerchief.
3) On discarded Cigarette Stubs.
4) On Cups, Tumblers, Bottles.
5) On Postage Stamps or Envelopes.
6) On Tooth Picks.
7) On piece of Cloth used as gag.
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Stains of saliva are not found on the crime related articles, as often as the stains of
blood and semen. In very peculiar contexts, however, their significance is high.
Unburnt portions of cigars, cigarettes or beedis are such examples. Saliva stain
grouping on such articles from the scene of crime is very helpful in the identification
of the criminal.1

HOW IS SALIVA DETECTED?

Saliva stains are detected by methods based on the presence of amylase. Amylase is
an enzyme that break down starch and glycogen into simple sugars. Amylase is found
in many body fluids ; very high in saliva.

TESTS

Two tests are there:

1. Starch iodine test
2. Phadebas reagent
A.) Press test
B.) Tube test

1Jaising P Modi, A Textbook of Medical Jurisprudence And Toxicology, LexisNexis, 25th ed., 2016,
page 523.
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STARCH IODINE TEST

It is one of the earliest method for saliva detection. It was devised by W. Roberts in
1881. In presence of iodine, starch turns blue. As amylase breaks down starch, the
colour changes and subsides.

PROCEDURE:

1. Wells containing different amounts of amylase are dipped in starch agar and
petri dish.

2. then wells are flooded with iodine solution.

3. if amylase (saliva) is present, the blue colour begins to vanish.

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DRAWBACKS OF THE STARCH IODINE TEST

1. The presence of proteins particularly albumin and gamma globulin originating

in other body fluids such as blood and semen, compete with starch for iodine
and produce false positive results.
2. The test is also difficult to use as a locator test for stains on items.2

PHADEBAS REAGENT TEST

WHAT IS PHADEBAS?

Phadebas is the synthetic biochemical substrate manufactured by pharmacia and

produced in tablet form.

It is used for both qualitative and quantitative assessment of the alpha- amylase
enzyme. Its active component is DSM-P, microspheres in which a blue dye has been
chemically bound.

When the substrate is digested by the amylase enzyme in solution, it releases that
blue dye at a rate proportional to the quantity of the enzyme present.

It is used in a range of applications from quality control tests for food and beverages
to detection of saliva stains in forensic investigations.3

2 D.K. Ganguly, Medical Jurisprudence And Toxicology, Dwivedi Law Agency, 2009, page 320-21.
3Dr. B.R. Sharma, Forensic Science in Criminal Investigation And Trials, Universal Law Publishing
Co. Pvt. Ltd., 5th ed., 2014, page 412.
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PHADEBAS REAGENT ( PRESS TEST)

The phadebas forensic press test is a new product that detects saliva stains by reacting
with amylase. When the paper is pressed against a saliva stain, a blue spot occurs.

PROCEDURE:

STEP 1: Place item to be tested on a flat and clean surface.

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STEP 2: Dampen Phadebas paper with water. Cover testing area with phadebas paper,
reagent side down.

STEP 3: Trace an outline of the testing area on the phadebas paper.

STEP 4: Cover the phadebas paper with a clean glass board. Weigh down the
arrangement. Paper is left on the area and watched for blue colour change.
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STEP 5: Start the timer and observe the test frequently during 40 minutes. record the
time of positive reaction ( distinct area of diffuse blue colour)

PHADEBAS REAGENT TUBE TEST

BACKGROUND

The tube test is more sensitive than the press test and used semi-quantitatively for the
presumptive testing of saliva deposits.

If it is suspected that the stain to be tested is a weak saliva stain, or if testing the
supernatant from an extracted stain or swab, Phadebas tube test offers a better method
than using Phadebas paper.

A tube test may also be carried out if amylase is detected (using the Phadebas
Forensic Press test) in an area that exhibits other staining such as semen, blood or
heavy vaginal deposits;
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The enzyme α-amylase is found in very high levels in saliva. Its activity in stains is
used as an indicator for the presence of saliva. The test used to identify amylase uses
Phadebas, consisting of starch microspheres with a blue dye cross-linked to the
starch. In the presence of amylase the starch is digested, releasing the water soluble
dye into solution. The resulting blue colour is read semi-quantitatively using a
spectrophotometer.


Although the Phadebas tests are specific for amylase , they’re not specific for saliva.
Amylase is found in other body fluids, although normally at much lower levels than
in saliva. Generally, amylase found in other body fluids will not be present in
sufficient quantity for detection by the Phadebas methods. As a reference on the
differences in amylase activity between saliva and other fluids, the below list was
compiled from the results published in reference 14:

Saliva: 263000 to 376000 IU/L

Urine: 263 to 940 IU/L
Blood: 110 IU/L
Semen: 35 IU/L
Nasal secretion: Undetectable levels
Sweat: Undetectable levels

When positive results are obtained on an item, it is recommended that a substrate

control sample also is submitted for DNA analysis at the same time, with the
exception of swabs.

PHADEBAS FORENSIC TUBE TEST PROTOCOL

If it is suspected that the stain to be tested is a weak saliva stain, or if testing the
supernatant from an extracted stain or swab, Phadebas reagent used as a tube test is

4 P.H. Whitehead, Ann E. Kipps; J. Forens. Sci. Soc. (1975), page 15, 39-42
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more sensitive than Phadebas paper. When testing swab(s) for the presence of saliva
they may need to be extracted according to a separate protocol. The supernatant from
the extraction procedure can then be tested using the Phadebas tube test protocol
below.

1. Prepare suitable positive (saliva) and negative (distilled water) control samples,
in tubes (for example Eppendorf tubes).

2. Extraction of samples: i. STAIN: Cut out a small portion of the stain (approx. 3x3
mm) and transfer to a sterile tube. Add 0.5-1 ml of sterile distilled water and
leave to soak for 1 minute. Agitate vigorously using a mechanical shaker for 30
seconds. ii. SWAB: Follow internal protocol for extracting swabs.

3. Pipette 0.5-1 ml of supernatant from the stain or swab extraction to another tube.

4. Add 1 (one) Phadebas tablet to each of the test and control tubes.

5. Top up the sample and control tubes to 1 ml with sterile distilled water and
agitate using a mechanical shaker.

6. Incubate the tubes at 37º C for 30 minutes. Remove and agitate each tube.

7. Centrifuge the tubes at 10,000 g for 1 minute.

8. A positive amylase reaction will produce a blue coloured supernatant solution,

the depth of colour depending upon the amylase concentration. A negative
reaction will result in a clear supernatant.5

5 http://www.phadebas.com/data/phadebas/files/document/
Instructions_Phadebas_Forensic_Tube_Test.pdf ; visited on 6th October, 2017 at 8:14 p.m.
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CONCLUSION

The identification of saliva stains may be important in the investigation of criminal

incidents. For example, a cigarette or handkerchief left behind at a crime scene may
have saliva stains present. If the person responsible for these stains is a “secretor”, it
may be possible to identify his blood group. This is also true for saliva stains on
postage stamps, the glued portion of envelopes, etc. DNA fingerprinting can be
carried out from saliva. Saliva per se does not contain body cells but it may well
remove sufficient epithelial skin cells from the inside of the mouth for DNA profiling
purposes. The technique has been shown to work well. Saliva does not give specific
precipitin test.