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Forensic Analysis of Diatoms in Drowning: Extraction and Procedure

Forensic Analysis of Diatoms in Drowning cases

Diatoms are living algae with a silica cell wall which are microscopic and unicellular. They are basically helpful in the diagnosis of antemortem drowning cases. So, for that, a proper forensic analysis is performed— from extraction to identification of diatoms.

Types of Samples Collected For Forensic Diatoms Analysis

In every diatoms analysis, two types of samples are collected:

  1. Postmortem Sample
  2. Reference Water Sample

1. Collection of Postmortem Samples

There are two types of postmortem samples that are analyzed simultaneously, in the examination of diatoms.

A. Bone Marrow From Long Bones

Sternum and Femur are most commonly used for collecting postmortem samples. This is because these two bones are founded to be the least contaminated.

Sternum and Femur are most common sample for bone marrow in for diatom analysis

However, if femur or sternum is not available, samples should be collected from tibia, humerus, radius, and ulna bones.

Bone Marrow Removal Technique For Diatoms
  1. Bones are washed with distilled water followed by the removal of the Periosteum (membranous tissue on the surfaces of bones).
  2. A rectangular piece of bone is removed.
  3. About 10gm of bone marrow is scooped up from the bone for diatom testing.

B. Collection of Soft Tissues

Soft tissues including the lungs and liver are majorly used for the collecting sample for forensic diatom analysis. 

Soft tissues are first washed with 1×1 cm of sample cut from the deeper tissues. This is because they are less contaminated as compared to the surface tissues.

However, some authors, instead of 1×1 cm of the sample, prefer to take at least 100g of the soft tissue sample.

In addition, in some cases where the chance of contamination is high, stream blood and brain tissues are also sampled for analysis.

2. Collection of Reference Water Sample

For reference, 500 ml of water from the suspected site of drowning. 

To the sample add 5 ml of iodine solution is added to kill any possible microbes. And then refrigerate the sample overnight. 

The reference water sample is analyzed to know whether the site is admissible to the finding of diatoms from the postmortem sample. This gives a clear understanding of whether or not the person is drowned at that suspected site.

Read More: Famous Cases Related to Diatoms: How Diatoms Helps in Solving Crime?

Extraction of Diatoms

For extraction of diatoms from postmortem and reference water samples, different methods are used. Let’s discuss them one by one.

A. Methods of Extraction of Diatoms at Postmortem

Methods of Extraction of Diatoms at Postmortem

There are majorly six diatoms extraction procedures. These are:

  1. Acid Digestion Method (mostly used)
  2. Enzymatic Digestion (mostly used)
  3. Indirect digestion of materials
  4. Ultrasonic Radiation (rarely use)
  5. Strong Anionic Detergents  (rarely use)
  6. Membrane filtering method (not in use)

1. Acid Digestion Method

The most common laboratory extraction for diatoms is the acid digestion method. 

Acid Digestion Method for diatoms

It used highly corrosive chemicals such as nitric acid and sulfuric acid. But majorly nitric acid is employed for the dissolution of rigid bones.

Procedure 
  1. 5g of marrow (or 100g of soft tissue) sample is taken in a beaker of 100ml. 
  2. Add 25 ml of conc. nitric acid to the marrow sample.
  3. The sample is left over for 2 days (dissolve all organic tissues)
  4. Centrifuge it for 30 minutes at 2000 to 3000 rpm.
  5. Now, the supernatant liquid is taken in a fresh flask leaving behind solid residue.
  6. To the solid residue, add 20 ml of water and centrifuge again with the above settings.
  7. Repeat steps 5 and 6 for 2 more times.
  8. Now, deposited nitric acid-resistant material is observed under a phase-contrast microscope.
Disadvantages of Using Acid Digestion Method
  1. Highly corrosive nitric acid required in a large amount.
  2. A large amount of tissue is required (100 g).
  3. Not effective for detection of soft tissue shells algae and phytoplanktons.
  4. Fats may not be dissolved properly.
  5. Very lengthy process.
  6. Repeated washing can also wash the diatoms away.

Though, the above are 6 main reasons not to perform acid digestion. But still, it is the most frequently used extraction method from tissue and water samples for diatom analysis.

2. Enzymatic Digestion Extraction For Diatoms

Enzymatic Digestion Extraction For Diatoms

Enzymatic digestion is considered to be more advantageous than acid digestion. It is a simple, rapid, and non-corrosive means of yielding diatoms. 

For enzymatic digestion, proteases such as proteinase K are used. This makes it a less destructive means of yielding even soft-bodied algae and protozoa that majorly get destroyed in acid digestion methods

So, using this extraction method, the examiner can effectively extract the soft-celled phytoplankton. 

Procedure
  1. 10g of soft tissues such as liver, lungs, brain, or femur bone marrow is ground/sliced with cuts.
  2. Add 500 ml of 10 mg/ml proteinase K.
  3. To the mix, add 0.01 M Tris HCl buffer (pH 7.5) containing 2% sodium dodecyl sulfate.
  4. Incubate the whole mixture at 50°C for overnight.
  5. Take the sample out and let it cool at room temperature.
  6. Now, add 100 ml of distilled water.
  7. Centrifuge it for 15 minutes at 3000 rpm.
  8. Discard the supernatant.
  9. Observe the deposit under a phase-contrast microscope

3. Indirect Digestion of Samples

Not commonly used, but it is one of the fastest procedures in the forensic examination of diatoms. Plus, it is the most destructive technique.

Procedure 
  1. Take 5 gm of sample and incinerate using an electric oven at about 300 to 400 °C.
  2. Powered the oven till all samples are incinerated and converted into ashes.
  3. To the ashes sample, digest it with 50 ml of nitric acid.
  4. The sample is centrifuged at 3000 rpm for 15 minutes.
  5. Discard the supernatant and add an equal amount of distilled water.
  6. Repeat it one more time.
  7. Take the dispositive and observe under a light microscope.

This method is very destructive and even destroys silica cells to some level. That’s why it is not recommended to perform an indirect digestion incinerated method for the extraction of diatoms.

Also Read:

4. Ultrasonic Radiation Extraction 

With the use of low frequency and high power ultrasound waves, the soft tissue samples or bone marrow are converted into solubilized tissues that are then used for microscopic analysis after acid digestion.

As these are majorly used to stabilize the soft tissue and then acid digestion is performed, it really takes an extra step but increases the digestion rate. Hence, less time is needed.

Though it takes less time, the efficiency of yielding a good quality of diatoms deposited for the observation is quite low.

A paper titled, “Effects of Ultrasonic and Microwave Pretreatment on Lipid Extraction of Microalgae and Methane Production from the Residual Extracted Biomass”, stated that the maximum lipid yield from B. braunii was 56.42% using microwave radiation and 39.61% for ultrasonication [Springer.com]

5. Extraction Using Strong Anionic Detergents 

Diatoms can also be extracted using strong anionic detergents. These detergents denature the protein and totally disrupted the membrane by breaking the protein linkage.

The most common strong anionic detergents are Sodium Dodecyl Sulfate (SDS) and Hydrogen peroxide.

It is a kind of enzymatic digestion because in most cases, it is employed in the process of enzymatic digestion. But still, it is a different technique. 

6. Membrane Filtration Method

A membrane filtration test is only used for the blood samples. It is a physical separation method that uses a filter funnel and vacuum system.

The apparatus is fitted with a special membrane to filter the molecules based on their size under an adjustable pressure (by vacuum system). 

Furthermore, due to contamination levels in the blood, blood is not currently viable for the diatoms analysis so is the membrane filtration method.

B. Method of Extraction of Reference Water Sample

For the extraction of water samples, majorly three techniques are employed. These are:

  1. Modified Acid Digestion Method (Fresh/Sea)
  2. Enzymatic Digestion Method

Please note: One should follow the same technique that they used for the extraction of diatoms from postmortem samples

1. Modified Acid Digestion Method (Fresh Water)

  1. Take the water sample in a 1000 ml beaker.
  2. Add 50 ml of concentrated nitric acid.
  3. Leave the sample mix for 2 to 3 hours.
  4. Centrifuged at 5000 rpm for 10 minutes.
  5. Discard the supernatant and take the residue pellets at the bottom.
  6. Add 30 ml of water and centrifuge it.
  7. Repeat it 2 more times.
  8. Residue pellets at the bottom are ready for observation.

2. Modified Acid Digestion Method For Sea Water

  1. Take the water sample in a 1000 ml beaker.
  2. Add 50 ml of concentrated nitric acid and boil the mix for 30 minutes in a water bath.
  3. Cool it at room temperature.
  4. Centrifuge the sample for 10 minutes at 3000 rpm.
  5. Washed the sample mix with ethanol (at least 2 times).
  6. Residue is heated and dried.
  7. Observe under an optical microscope. 

3. Enzymatic Digestion Method 

  1. To the water sample, add hydrogen peroxide (130 vol%).
  2. Heat the mix for 12 hours at 80°C.
  3. Cool the mix at room temperature.
  4. Centrifuge it for 15 minutes at 2500 rpm.
  5. Discard the supernatant, and dilute the residue again.
  6. Repeat steps 4 and 5 for two more times.

Procedure For Diatom Test 

The following are the procedure to perform the diatom test in cases of drowning:

  1. Take a small amount of residual material from the extraction, and make at least 5 to 10 slides (for more possible findings).
  2. The residual material is allowed to dry on the slide so that it gets fixed.
  3. And make it permanent using a drop of DPX mountant on each of the slides.
  4. Cover the slide with a coverslip and observe under a microscope.

The same procedure is used for the forensic analysis of the water reference sample that is used for diatoms.

Please Note: DPX mountant is a synthetic transparent mounting media that is used to fix the sample on the slide by quickly drying over it.

Observation and Result

Requirements: 

  • Preference 1: Phase Transfer Microscope 
  • Preference 2: Compound Light Microscope on 1000x 

Result 1: Positive Diatom Test

Positive Diatom Test signifies

If the control sample and postmortem sample has the same algae structural (type, size, and density) uniqueness then it is a positive sign of antemortem drowning at the site from which the reference sample is collected.

Result 2: Negative Diatom Test Result

Negative Diatom Test Result signifes

A negative diatom leads to three common scenarios:

  1. Neither of the samples (postmortem and reference sample) has diatoms.
  2. One sample has diatoms but others don’t
  3. Both have diatoms but of different structural

A. Neither of the samples have diatoms

This indicates that the death is not due to drowning because the person is unconscious when he/she was drowning.

B. One Sample has Diatoms but Others Don’t

Situation 1: If the postmortem sample has diatoms and a reference water sample don’t

Death is due to drowning but the body is moved to a new water source. However, it is fairly unlikely, because most natural water bodies have diatoms.

Situation 2: If the reference water sample has diatoms but the postmortem sample doesn’t.

Death is not due to drawing. The body is sacked down after the person becomes unconscious. 

DiatomsPost Mortem Sample Water SampleFeatures
Present/AbsentPresentAbsentAntemortem Drowning 
Present/AbsentAbsent PresentPost mortem drowning

C. Both have Diatoms but of Different Structure

In that case, the death is due to antemortem drowning but the body is moved from its original source. 

This can be because of:

  • Deliberately for miss guiding the case, or
  • Body moves with the water flow and comes to the surface after 3 to 4 days because of putrefaction. 

General FAQ

1. Why does nitric acid doesn’t disrupt the diatoms but tissues only?

First of all, nitric acid is highly corrosive that digests almost all the biological tissues. But fail to disrupt the diatoms because they have silica-based unique cell walls (frustule).

2. What does a diatom result indicate?

If the post mortem sample test has a diatom (or positive diatoms result), it means the death is due to antemortem downing. However, if the test is negative, the death is because of other means such as any trauma, medical condition, poison, etc.

Reference

  • http://www.cambridge.org/9780521363181[Cambridge]
  • https://www.sciencedirect.com/science/article/abs/pii/0300943272900155 [ScienceDirect]
  • https://pubmed.ncbi.nlm.nih.gov/9068187/[PubMed]
  • https://www.researchgate.net/publication/15198886_Application_of_a_simple_enzymatic_digestion_method_for_diatom_detection_in_the_diagnosis_of_drowning_in_putrified_corpses_by_diatom_analysis [ResearchGate]
  • https://pubmed.ncbi.nlm.nih.gov/11197630/[PubMed]
  • Forensic Medicine & Toxicology, Book by Anil Aggrawal

Further Readings:

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