Diatoms

Diatoms are microscopic unicellular, photosynthetic, eukaryotic algae that inhabit almost all bodies of water.

They are found in springs, rivers, ponds, lakes, ditches and in freshwater and marine waters and occur in terrestrial habitats such as wet rocks, mosses and soils, even caves.

CLASSIFICATION

Domain – Eukaryota

Kingdom- Chromalveolata

Phylum- Heterokontophyta

Class –Bacillariophyceae

Orders- Centrales & Penales

Diatoms have traditionally been classified according to the shape of their valves, divided into two major structural groups:

• Centric Diatoms (With valves exhibiting a radial symmetry)
• Pennate Diatoms (With valves exhibiting bilateral symmetry)

IMPORTANCE OF DIATOM IN FORENSICS

When a dead-body is recovered from water, there is always a question whether it was a case of ante-mortem or post-mortem drowning i.e. whether the body was drowned before or after death. In these medico legal cases, presence of diatoms in the body tissues is very useful evidence.

It helps to differentiate a death by submersion from an immersion of a body.

Laboratory tests may reveal the presence of diatoms in the body.

Their silica-based skeletons do not readily decay and they can sometimes be detected even in heavily decomposed bodies.

If the person is dead when entering the water, then there is no circulation and the transport of diatom cells to various organs is prevented because of a lack of circulation and diatoms cannot enter the body.

If the person is still alive when entering the water, diatoms will enter the lungs if the person inhales water and drowns. The diatoms are then carried to distant parts of the body such as the brain, kidneys, and bone marrow by circulation.

Since diatoms resist putrefaction, the diatom test is particularly valuable, where decomposition is advanced. Diatom test is negative in dead bodies thrown in water and in dry drowning.

Forensic Limnology

Forensic limnology is a sub-field of forensic botany. this field mainly examines the presence of diatoms in crime scene samples and victims.

TECHNIQUES AND METHODS

Materials taken from victims/suspects/crime scene or Environmental products, i.e. mud

• Keep in a Test tube with a sulfuric acid and potassium dichromate solution for 24 hours.
• Centrifuge The Tube.
• Placed in A boiling water bath for an hour.
• Pellet Is Extracted.
• Mixed with distilled water.
• mounted on a slide with Hyrax.
• Use phase contrast microscopy for observing Diatoms.

Diatoms have to be cleaned before examination so that any foreign material can not interfere with microscope examination.

The use of diatoms as a diagnostic test for drowning is based upon the hypothesis that diatoms will not enter the systemic circulation and be deposited in such organs as the bone marrow unless the circulation is still functioning thus implying that the decedent was alive in the water.

for the detection and examination of diatom , Hard bones (sternum and femur) and soft tissues (lungs and liver etc.) of drowned bodies are usually sent to the Forensic Science Laboratories.

SOLUENE-350 METHOD

It’s a faster and less expensive Test. The Soluene-350 method is used only for freshwater samples.

• The sample should mixed with formalin, rinsed and centrifuged three times with distilled water. 
• The pellet should removed from the test tube added with 8ml of Soluene-350.
• After that it should Incubated at 50 degrees Celsius for 2 hours
• Then it should Centrifuged again.
• After all the process it should be Mounted on a slide to see under microscope.

ENZYMATIC METHOD

The ‘’’Enzymatic method’’’ is used for tissue samples.

Because of the use of enzyme chemicals it has a smaller percentage of damaging the diatoms collected than acid digestion method.

• In this method Concentrated Nitric acid and Proteinase-K, or peroxide, should add to the sample for 12 hours,
• The sample should be Centrifuged Twice before further process.
• After that it should Rinsed with distilled water
• Then it should Mounted on a slide with Naphrax
• After all the process use a light microscope when examining the results.

COLLOIDAL SILICA GRADIENT CENTRIFUGATION METHOD

• Colloidal Silica Gradient Centrifugation method can be used with liver and lung samples.
• The collected tissue is added to saline solution, centrifuged at a temperature of 12 degrees Celsius.
• Then distilled water should be distilled water
• Then it should be mounted on a slide and observed.

MEMBRANE FILTER METHOD

• The ‘Membrane Filter method’ is used when there is a presence of either damaged diatoms in the sample (result of pollutants) or inorganic materials.
• The blood of the victim should be taken and filtered through Sodium Dodecyl Sulfate and nitrocellulose membranes with decreasing pore sizes.
• The membranes will be dry and filter with distilled water.
• After being dried the pieces of membrane that filtered the blood, should be examined for diatoms and pollen grains.

DRY ASH METHOD

‘Dry Ash method’ can only be applied to a bone marrow sample.

Five grams of marrow should be taken from a victim to be placed in a nitric acid solution and then burnt in a furnace allowing the release of diatoms to then be analyzed. whether the body was drowned before or after death. In these medico legal cases, presence of diatoms in the body tissues is very useful evidence.

NITRIC ACID METHOD

• Samples should be collected from the suspected drowning victim. Care should be taken as to not contaminate the sample with foreign diatoms during the process.
• For example, Intact femurs are removed at autopsy and washed in distilled water. Femurs are longitudinally sectioned using a clean band saw, and the bone marrow about 50g is removed using a clean spatula and placed into a boiling flask.
• Approximately 50 mL of conc. nitric acid need to be added to the flask, and the marrow-acid suspension will be boiled on a hot plate for approx 48 hours-under a fume hood.
• The suspension should be cooled and centrifuged, in some instances at two different times, the supernatant should be discarded and the resulting acid-resistant material dropped onto a clean microscope slide and the sediment examined under the microscope Should be done.

SULPHURIC ACID METHOD

• First all calcareous compounds have been removed so that the sample will not form gypsum crystals.
• When sample has settled completely then discard supernatant.
• Add conc. sulphuric acid until the volume is twice.
• Just add enough Potassium bichromate to make for a saturated solution.
• Let stand for 24 hours or more, or speed up the reaction in a water-bath 60 degrees. The sediment should look grayish.
• Let settle completely, discard supernatant and rinse several times.

ELECTRON MICROSCOPY

Electron or dark phase microscopy is currently the main methods used for analysis. In order to examine the morphology of diatoms, both transmission and scanning electron microscopes are able to provide a much more detailed image. These microscopes were necessary for taxonomic purposes, with the distinctions between species being so minute at times.

Scanning Electron Microscopy (SEM)

SEM is best for visualizing the entire diatom Frustule. It is a device that can visualize the gross morphology of the diatom’s both internal and external parts.

Transmission Electron Microscopy (TEM)

This type of microscopy is best. with the help of this device we can see the finer, delicate details of the diatom frustule.