Test 3: Microscopy, DNA, Serology

Microscopy

Microscopy

Micro = small

Skopein = to see

Janssens (1590)

• Dutch spectacle makers

• Discover that nearby objects greatly enlarged with lenses

Galileo (late 1600s)

• Created a better device with a focusing instrument based on the Janssens’ experiments

Other Early Microscopes

• Hooke Microscope (1670)

• Leeuwenhoek Microscope (late 1600s)

Later Microscopes

• Pacino (1870)

• Olympus (Modern)

Parts of a Light Microscope

Total Magnification

Objective lens x eyepiece (10x)

• Ex: 4x objective x 10x eyepiece = 40x total magnification

Fixation

Chemically preserve specimen

Stains

Preparation of specimens for light microscopy

Differential Staining

• Differential stains react differently with different kinds of bacteria in order to distinguish them

• Gram stain divides bacteria into two large groups, gram-positive and gram-negative

Comparison Microscope

• Important tool to make side-by-side comparisons

• Two compound microscopes combined into one unit

• When the viewer looks through the eyepiece, a field divided into two equal parts is observed

Polarizing Microscope

• Based on the rotation of polarized light

• Polarizing potato starch

Fluorescence Microscope

• Specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near ultraviolet) light source

• The microorganisms appear as bright objects against a dark background

• Used primarily in a diagnostic procedure called fluorescent-antibody (FA) technique, or immunofluorescence

Electron Microscope

• A beam of electrons, instead of light, is used

• Magnifies greater because the wavelengths of electrons are much smaller than those of visible light = 0.005nm as opposed to 500nm (100,000x smaller)

• The best compound light microscopes can magnify 2000x, while electron microscopes can magnify up to 100,000x

Scanning Electron Microscope

• Scanning electron microscopes (SEM): 3D views of the surfaces by aiming a beam of electrons into the specimen

  • Electrons are bounced off the surface of the specimen and form a 3D image that is stereoscopic in appearance

  • Magnification: 1000-10,000x, and Depth of Field very high

  • Can be used to identify the elements present in the specimen under examination

Nucleotides

• The monomers that form the polymer of DNA

• Each consists of:

  • Deoxyribose

    • Pentose sugar

  • Phosphoric acid

  • A nitrogenous base

Double Helix

• Double-stranded macromolecule

• Two polynucleotide chains are held together by H-bonds

  • A(denine) always pairs with T(hymine)

    • two H-bonds

  • C(ytosine) always pairs with G(uanine)

    • three H-bonds

“Anti-parallel Molecule”

• In a double helix, the strands go in opposite directions

• 5’ = free phosphate group (top)

• 3’ = free OH group (bottom)

Nuclear DNA

In the nucleus

Mitochondrial DNA

In the mitochondria

• Self-replicating cell

• Only DNA of the mother

• Because of the way that the mitochondria exist in the sperm, the mitochondria in the sperm don’t become part of the fetus

• >1000 copies/cell

• Maternally inherited

• Not unique to individual

• Circular like bacteria

Polymerase Chain Reaction

• Simulated natural DNA replication

• Copy SECTIONS of DNA, not the whole molecule

  • Only the 0.1% that differs between people

RFLP Technique (1980s)

Restriction Enzymes - cut DNA

Probe - radioactively charged DNA that searches for other DNA

1. DNA samples with added restriction enzymes produce restriction fragments

2. Electrophoresis separates the restriction fragments. Each sample forms a characteristic pattern of bands

3. Alkaline solution is pulled upward through the gel to a sheet of nitrocellulose laid on the top of it, transferring the DNA to the paper

4. The paper is exposed to a solution containing radioactively-labeled probe

5. The photographic film laid on top of the paper is exposed to the radioactivity in the bond probe to form an image corresponding to the DNA bands

   1. The probes are looking for Variable Number Tandem Repeats (VNTR)

Variable Number Tandem Repeats (VNTR)

• Can contain anywhere from 20 to 200 base pairs

• In Intron region of DNA (not Exon region)

• Inherited from mom and dad

Short Tandem Repeats (STR)

• 13 CODIS core STR loci with chromosomal positions

• Areas of repeating sequences

  • AGAT on C5

STR DNA Analyzer

• Have become more advanced

• DNA enters on end of the machine, and a profile is produced from the other

DNA Fingerprint

• Peaks show the repeats of sequences

CODIS - Combined DNA Index System (1998)

• Links to serial crimes and unsolved with repeat offenders

• Interstate FBI database

• Requires >4 RFLP markers and/or 13 core STR markers

• Current backlog of >600,000 samples

DNA Chips

Small chips that will make copies of DNA at the crime scene

Geotracking

Swabbing objects can reveal where its been based on the DNA of plants, animals, and fungus found on it

Serology

the study of blood

Blood

• A complex mixture of cells, enzymes, proteins, and inorganic substances

  • Fluid portion of blood is called plasma (55%)

    • Primarily water

  • Cells

    • Red cells (erythrocytes)

    • White cells (leukocytes)

    • Platelets (thrombocytes)

Plasma

Contains fibrinogen, salts, proteins, glycoproteins, carbohydrates, antibodies, hormones (insulin, testosterone, estrogen, adrenaline, norepinephrine, etc.), albumin, and dissolved gases

Red Blood Cells (erythrocytes)

• Most abundant cell

• Average of 5,000,000 RBCs per cubic microliter

• Accounts for 40-45% of the blood

• Population measured with a hematocrit

• The ratio of cells in normal blood is 600 RBCs for each WBC and 40 platelets

Presumptive Test

• An analysis that suggests blood could be present

• Fast and relatively sensitive

Confirmatory Test

An experiment that can indicate the presence of blood with a high degree of certainty

Benzidine Color Test

Dropped due to carcinogenic reagents

Kastle-Meyer Test

• Uses phenolphthalein to turn blood a deep pink color

Luminol Test

• Reaction of luminol with blood produces a complex which can be seen by luminescence

• Very sensitive - up to a 3,000,000 dilution of blood can be seen

• Detects unseen samples and patterns

• Does not interfere with later DNA testing

Precipitin Test

• Blood origin test

• When animals are injected with human blood they form antibodies to the human blood. This can isolate human antiserum (antibodies to human blood)

• Human antiserum will react with human blood

• Antiserum can (has) been made similarly for many animals

• Works on old (years dried) and small samples

Karl Landsteiner (1900s)

• Noticed that a mixing of blood sometimes forms a precipitate

• A, B, AB, O, and Rh proteins

Transfusions

A: anti-B plasma antibodies (42%)

B: anti-A plasma antibodies (12%)

AB: person has no plasma antibodies against blood antigens (3%)

• Universal receiver

O: anti-A and anti-B antibodies (43%)

• Universal donor

Paternity

Find with a punnet square

Determination from Nonblood Fluids

• 80% of people are Secretors

• Find blood antigens (ie A, B, Rh) in saliva, semen, vaginal fluids, gastric juice

Passive Bloodstains

• Created or formed solely by the force of gravity (low speed)

  • Force of impact is 5ft/sec or less

  • Size of the droplets between four and eight millimeters (0.16 to 0.31 inches)

  • Can be subdivided into drops, drip patterns, pools, and clots

Target Surface Type

• Bloodstains can occur on a variety of surfaces including clothing, carpeting, walls, etc.

• The type of surface the blood strikes affects the nature of the observed splatter

  • Hard smooth surcace (eg glass): little distortion around the edges of the droplet

  • Irregular linoleum flooring: often show distortion (scalloping) around the edge of the droplets

  • Wood or Concrete: distorted to a larger extent (eg spines and segondary splatter)

Drip Pattern

• free-falling drops dripping into wet blood

• Large irregular central stain

• Small round and oval satellite stains

Transfer Bloodstains

created when a wet, bloody surface comes into contact with another surface

• Ex: contact bleeding, swipe, wipe, and smudge

Projected Bloodstains - Impact

Created when a blood source is subjected to an action greater than the force of gravity

• Medium Velocity - force of 5 to 25 feet/sec

  • Stain size 1 to 4 mm

  • Beatings, blunt object trauma

• High Velocity - force of 100 feet/sec

  • Stain size about 1 mm and smaller

  • Misty appearance

  • Gunshot

Other Projected Blood Patterns

• Arterial Spurts (Vertical and Horizontal)

  • Bloodstain pattern from blood spurt under pressure from a cut artery

Point of Convergence and Origin Determination

• By drawing a line through the long axis of a group of bloodstains the point of convergence can be determined

• Where the lines of the group of stains intersect one another the convergence point can be established

• Directionality is usually obvious as the pointed end of the bloodstain (tail) will always point in the direction of travel

• By drawing a line through the long axis of a group of bloodstains the point of convergence can be determined. Where the lines of the group of stains intersect one another the convergence point can be established

Angle of Impact

sin(theta) = (width/length)