Forensic Biology Screening Workshop
Other Body Fluids and Tissues
Vaginal Secretions
Vaginal Secretions
• Vaginal secretions are a complex mixture of cells
and secretions
• There is no confirmatory test to identify vaginal
secretions
• Several screening tests based on microscopy
have been proposed.
Vaginal Secretions
• Vaginal epithelial cells are large, and many
contain glycogen (a polysaccharide) which can
be demonstrated by staining with iodine in the
form of a solution or exposing to iodine vapor.
• The presence of glycogenated cells is variable
depending on the stage of the estrous cycle
• Glycogenated cells can be found in other body
secretions (i.e. oral, anal)
Vaginal Secretions
• Based on the very high glycogen content of the
vagina, vaginal secretions have a higher
quantity of glycogenated cells than other body
fluids
• Lugol's solution, first made in 1829, is a solution
of iodine
– Named after French doctor, Lugol
Lugol’s Staining
• Squamous epithelial cells
with high glycogen
content will show a
chocolate brown color in
the body of the cell
(cytoplasm) with a clear
round to oval shaped
unstained nucleus
located somewhat
centrally in the cytoplasm
Lugol’s Staining
HOW TO MAKE:
– Iodine (0.02m) 1 g
– Potassium iodide (0.06m) 2 g
– Distilled water 200 ml
Dissolve the potassium iodide in the water, then
add iodine.
HOW TO STORE:
– Relatively stable at room temp or 4C
Lugol’s Staining
HOW TO PERFORM:
• Make a thin smear of the questioned material on
a glass microscope slide
• Fix the smear by gentle heating
• Cover the smear with Lugol’s iodine solution
• Add an equal volume of water to the smear
Lugol’s Staining
HOW TO PERFORM:
• Place a cover slip on the stained area
• Allow to stand 3-5 minutes at room temperature
• Observe microscopically at 200-400x
Lugol’s Staining
HOW TO PERFORM:
• Squamous epithelial cells with high glycogen
content (notably vaginal and penile urethral
epithelial cells) will exhibit a chocolate-brown or
tan color
• Other epithelial cells will exhibit a yellow or gold
color
Lugol’s Staining
HOW TO PERFORM:
• A majority of epithelial cells stained chocolate
brown is a positive result.
• Known vaginal cells should be tested as a
positive control
• Known buccal cells should be tested as a
negative control
Lugol’s Staining
epithelial cells stained
chocolate brown
•Gold/yellow stained
epithelial cells
Fecal Material
Fecal Material
• Feces are food residues passed after
completion of travel through the digestive
system
• Has a characteristic odor mainly due to skatole,
an organic compound that occurs naturally in
feces
Fecal Material
Microscopy
• Microscopy has been used to identify fecal
material
– Looking for undigested residues of food
material
Chemical Tests
• Detection of urobilinogen, a bile pigment
excreted in feces, which may be detected using
its fluorescent reaction to Edelman’s reagent
Test for Urobilinogen
HOW TO MAKE:
• Solution #1: 40% Alcoholic mercuric chloride solution
– Mercuric chloride 4 g
– Methanol 10 ml
Mix and store in stopper bottle
• Solution #2: 40% Alcoholic zinc chloride solution
– Zinc chloride 4 g
– Methanol 10 ml
Mix and store in stopper bottle
• Solution #3: Amyl alcohol
Test for Urobilinogen
HOW TO STORE:
– Relatively stable at room temp or 4C
Test for Urobilinogen
HOW TO PERFORM:
•
Extract the suspected stain and an unstained control in
3 drops of water in separate test tubes
•
Place an equal amount of water in a third test tube for
the negative control
•
Extract a known stain in 3 drops of water
•
Add 3 drops of 40% alcoholic mercuric chloride to each
tube
Test for Urobilinogen
HOW TO PERFORM:
• Add 3 drops of amyl alcohol to each tube. Shake to mix
• Centrifuge and collect the clear supernatant
• Examine under UV long-wave light for any fluorescence
No fluorescence should be visible at this point in the
analysis
• Add 3 drops of 40% alcoholic zinc chloride to each tube
and shake
Test for Urobilinogen
HOW TO PERFORM:
•Incubate at room temperature for 30-60 minutes
•Examine under long-wave UV light. The
appearance of a green fluorescence is considered
a positive test for urobilinogen, indicative of the
presence of fecal matter
Test for Urobilinogen
HOW TO PERFORM:
•Known fecal material should be tested as a
positive control
•Water should be tested as a negative control
Note: Safety eyeglasses which absorb ultraviolet
radiation must be worn when examining for
fluorescence with UV light
Urine
Urine
• No confirmatory tests for the presence of urine
• Urine stains fluorescent under ultraviolet light
– This can be useful for locating stains prior to chemical
testing
• Has a characteristic odor
• Contains a large amount of urea, a chemical byproduct
of normal metabolic processes in the body
– Identification of high levels of urea can serve as a
screening test for urine in fluids or stains
– Perspiration can give reactions similar to urine
Urine
• Contains creatinine, which is a breakdown
product of creatine (an important part of muscle)
– Over time, the creatine molecule gradually
degrades to creatinine
– Creatinine is a waste product that is excreted
from the body entirely by the kidneys
– Identification of creatinine can serve as a
screening test for urine in fluids or stains
Urea-Litmus Paper Test
Litmus Paper test for the detection of ammonia
•Relies on the indirect identification of urea by
reacting a test sample with urease to generate
ammonia from the urea. Litmus paper is used to
detect the ammonia
Urea + H2O ↔ CO2 + 2NH3
•A known urine sample and a blank are tested as a
positive and negative control. A substrate control
may be tested, as needed
Urea-Litmus Paper Test
•Affix a strip of litmus paper to a cork or rubber
stopper that will fit into the test tubes used: cut a
slit in the bottom of the stopper and insert one end
of the paper. The paper strip should hang down
into the tube without touching the sides of the tube
or becoming wet
•Place a cutting from a suspected urine stain in a
test tube with 3-4 drops of distilled water
•Add a drop of urease solution (urease mixed with
distilled water) to each tube
Urea-Litmus Paper Test
• Incubate at 37°C for 30 minutes
• A definite and easily observed color change to
blue on the litmus paper represents a positive
result
Urea-Nitrogen Tube Test
• Relies on the indirect identification of urea by
reacting a test sample with urease to generate
ammonia from the urea. Ammonia is
subsequently identified by the production of a
deep blue-colored solution
Urea + H2O ↔ CO2 + 2NH3
Urea-Nitrogen Tube Test
HOW TO MAKE:
• A urease buffer solution
• A urea-nitrogen standard solution
• A phenol-nitroprusside reagent
• An alkaline hypochlorite reagent
Urea-Nitrogen Tube Test
HOW TO STORE:
• The urease buffer solution is not stable at 4° C.
for more than a few weeks. Make fresh solution
according to concentration stated on label
(30 mg urea/100 ml distilled water)
• Other reagents-see product inserts
Urea-Nitrogen Tube Test
•A known urine sample and a blank are tested as a
positive and negative control. A substrate control
may be tested, as needed
Urea-Nitrogen Tube Test
Step 1
Sample
Negative Control
Substrate
1 drop H2O
Enzyme
0.5 ml urease
Standard
1 drop ureanitrogen standard
0.5 ml urease
Positive Control
Appx 1 cm2 stain
0.5 ml urease
Substrate Control Appx 1 cm2
substrate material
0.5 ml urease
Question Sample Appx 1 cm2
(without urease) unknown stain
Question Sample Appx 1 cm2
unknown stain
none
0.5 ml urease
Urea-Nitrogen Tube Test
• Cover tubes and allow to stand 20 minutes at
room temperature
• Add 1.0 ml phenol-nitroprusside reagent to each
tube
• Add 1.0 ml alkaline hypochlorite reagent to each
tube
• Invert to mix. Read results within 5 minutes.
Urea-Nitrogen Tube Test
Sample
Expected Results
Negative Control
No color
Standard
Deep blue color
Positive Control
Deep blue color
Substrate Control
No color
Question Sample (without urease) No color
Urea-Nitrogen Tube Test
Question Sample
• If stain is urine, a deep blue color should be observed
which should be as intense as the urine positive control.
• If the color is not as intense as the urine positive control
or the color in the question sample (without urease) tube
is as intense as the sample tube, this constitutes an
inconclusive test result
• If no color is observed, this is a negative result
Creatinine
Jaffe Test
• One of the oldest tests for the detection of
creatinine-1886
• Creatinine forms a red compound with picric
acid (Jaffe test)
Jaffe Test
HOW TO MAKE:
• Saturated aqueous picric acid solution
– 1 gram picric acid in 30 ml H2O
• 5% Sodium hydroxide
• Glacial acetic acid
Jaffe Test
HOW TO PERFORM:
• Cut a portion of the suspected stain and an
unstained control area and place on filter paper
• Cut a portion of a known urine stain and place
on a separate filter paper
• To each, add 1 drop of saturated picric acid
solution followed by 1 drop of 5% NaOH
Jaffe Test
• An orange coloration after 15 minutes on the
questioned stain and surrounding filter paper is
considered a positive result. The control area
should remain yellow.
• Addition of 2 drops of glacial acetic acid should
render the orange questioned stain yellow (decolorization), further confirming the presence of
creatinine
HAIR
Hair
• Composed of cylindrical structures or shafts
made up of tightly compacted cells that grow
from follicles
• Diameter ranges from 15-120 µm
– Depends on type of hair and body region
• Root material can be used for nuclear DNA
testing
• Shaft material can be used for mtDNA testing
Hair
• Human hairs are distinguishable from hairs of other
mammals
– Human hairs are generally consistent in color and
pigmentation throughout the length of the hair shaft,
whereas animal hairs may exhibit radical color
changes in a short distance, called banding
– The medulla, when present in human hairs, is
amorphous in appearance, and the width is generally
less than one-third the overall diameter of the hair
shaft. The medulla in animal hairs is normally
continuous and structured and generally occupies an
area of greater than one-third the overall diameter of
the hair shaft.
Hair
Structure
• Three cell types
– Outer cuticle
– Central cortex
– Central medulla
Hair
• In some instances human hairs can be classified by
racial origin such as Caucasian (European origin),
Negroid (African origin), and Mongoloid (Asian origin).
• In some instances the region of the body where a hair
originated can be determined by its gross appearance
and microscopic characteristics
• The length and color can be determined
• It can also be determined whether the hair was forcibly
removed, damaged by burning or crushing, or artificially
treated by dyeing or bleaching.
Hair
The growth phase of the hair is important in
determining whether the root is suitable for nuclear
DNA analysis testing
•Growth Cycles
– Anagen phase
– Catagen phase
– Telogen phase
Hair
Anagen Phase
• Active hair growth
• Contains nucleated cells in the root and in the
surrounding sheath material
• Generally suitable for nuclear DNA analysis
Hair
Anagen Hair
Hair
Catagen Phase
• Transitional phase after active hair growth, cell
division stops
• Characteristic club appearance of root
• May be suitable for nuclear DNA analysis
Hair
Catagen Hair
Hair
Telogen Phase
• Follows transitional phase-growth ceases
• Shedding phase
• Telogen hairs without follicular tissue may not be
amenable to nuclear DNA analysis because of
the lack of nucleated cells
– May contain sufficient mitochondrial DNA in
their roots and hair shafts for analysis
Hair
Telogen Hair
Hair
Basic Evaluation Steps
1. Determine if the sample is a hair
2. Determine if the hair is of human origin
3. Determine if the hair has root material-suitable
for nuclear DNA analysis (Characteristic of a
particular growth phase )
4. If not suitable for nuclear DNA analysis,
determine if the hair is sufficient in size for
mtDNA analysis (2-3cm)
Hair
DNA analysis of hair is a destructive technique and
results in the consumption of portions of the hair
– Hair characteristics, such as color, length,
shape, and texture should be noted in the
case file for future reference prior to DNA
analysis
• Notes and digital images
Hair
Hair Comparisons
• There are instances where useable nuclear DNA
will not be extracted from a hair and comparison
microscopy may be the only way to provide an
association of the questioned hair to known
hairs
– The laboratory’s procedures should address
whether microscopical hair comparisons are
performed prior to DNA analysis
Hair
Hair Comparisons
• There will be instances when mtDNA may not provide
adequate discrimination among individuals. In these
cases, a microscopical examination might provide
sufficient discrimination of the hair to associate a
questioned hair to a known hair
– Most laboratories performing mtDNA analysis have
procedures that require microscopical hair
comparisons be performed prior to mtDNA analysis
– A combination of mitochondrial DNA and comparison
microscopy may help to exclude or provide a stronger
association than the use of either technique alone