Diatoms: Forensic Significance and Analysis

Diatoms: Forensic Significance and Analysis

Diatoms are microscopic algae with silica cell walls that are commonly found in aquatic environments. In forensic investigations, diatoms play a significant role in determining whether drowning occurred before or after death.

Objectives

• Forensic Significance of Diatoms

• Sample Collection

• Acid/Chemical Digestion Method

• Enzymatic Methods

• Microwave Digestion, Vacuum Filtration, and Scanning Electron Microscope

• Soluene 350 Digestion

Forensic Significance of Diatoms

Sample Collection

• Bone marrow (sternum or femur), lungs, liver, kidneys

• Water samples from suspected drowning site

Acid/Chemical Digestion Method

• The most common and oldest form of method is acid digestion or chemical digestion of samples.

• This method has been used globally and has proven successful in recovering intact diatom frustules from the samples.

Reagents Used

• Nitric acid (HNO₃)

• Hydrochloric acid (HCl)

• Hydrogen peroxide (H₂O₂)

• Sulfuric acid (H₂SO₄)

Procedure

1. Thin strips of tissues, namely lungs, liver, and kidney, along with the water sample were separated in a flask and allowed to boil by using distilled water.

2. 10 ml of HNO₃ with 30% H₂O₂ was added to the samples, and the sample acid mixture was boiled carefully.

3. This mixture was allowed to cool and washed with distilled water after frequent centrifugations at 3000 rpm.

4. The final sediment was then analyzed microscopically.

Enzymatic Methods

Procedure

1. Tissues were treated with 500 μl of proteinase K (10 mg/ml), 100 ml of 0.01M Tris-HCl buffer solution (pH 7.5), and 2% SDS.

2. The mixture was incubated at 50 °C and left undisturbed.

3. The sample mixture volume was diluted using 100 ml distilled water, followed by centrifugation at 3000 rpm for 15 minutes.

4. The final residue was analyzed microscopically.

Note

The digestion of the sample was done by using Qiagen Proteinase K, Qiagen Buffer ATL, and 5 NHCl.

Limitation

The enzymatic method was found to be better than chemical digestion, but its major drawback is that it is costly.

Microwave Digestion, Vacuum Filtration, and Scanning Electron Microscope

Procedure

• 20 ml water along with 2 g of thin strips of lungs, liver, and kidneys were digested in microwave MW000 with the addition of 6 ml of HNO₃ and 2 ml of H₂O₂.

• The sample acid mixture was allowed to liquefy by increasing the microwave power for 5–10 minutes.

• The fluid was further subjected to vacuum filtration, and the results were automatically scanned using a Scanning Electron Microscope (SEM).

Compared to previously discussed methods, this method proved more efficient, rapid, and safer with a lower chance of contamination.

Soluene 350 Digestion

Procedure

• Take sample (5 g).

• Transfer the sample into a 30 ml glass tube followed by the addition of Soluene 350.

• The mixture is placed in an ultrasonic cleaner with full exposure to ultrasonic waves.

• The sample is then centrifuged at 3000 rpm for 5 minutes.

Creating Permanent Slide of Diatoms

The permanent slide of the sample is made by:

• Mounting the material with a high refractive index mountant, such as:

  • Naphrax (~1.74)

  • DPX (~1.520)

  • Euparol (~1.483)

• A coverslip is placed over the sample, and the slide is allowed to dry to fix the sample permanently.

Diatoms: Forensic Significance and Analysis