Method for extracting diatom in lung of drowning corpse in turbid water body

Abstract

The invention discloses a method for extracting diatom in drowning corpse lung in a turbid water body, which comprises the following nine steps: determining the density of a diatom skeleton contained in a specific water body and the density of a siliceous impurity skeleton, preparing a heavy liquid, carrying out microwave digestion on lung tissue, carrying out vacuum suction filtration (I), carrying out ultrasonic desorption, centrifuging, extracting a diatom heavy liquid, carrying out vacuum suction filtration (II) and coating. The impurity interference is reduced, and the diatom recovery rate and the diatom identification accuracy are improved. The skeleton density of the diatom and the siliceous impurities contained in the specific turbid water body is obtained by means of digestion, freeze-drying, gas replacement method true density instrument measurement and the like for the first time, and the heavy liquid which is used for separating the diatom and is appropriate in density and economical is accurately prepared according to the skeleton density. The problems of low diatom extraction rate and inaccurate classification due to interference of siliceous impurities during detection of drowning carcass lung diatom in a turbid water body are effectively solved. The method is of great significance in improving the value of diatom inspection in drowning diagnosis and drowning place inference.

Description

Technical Field

[0001] The invention relates to the field of forensic medicine examination, and in particular to a method for extracting diatoms in the lungs of drowned corpses in turbid water. Background Art

[0002] Analyzing the cause of death of bodies in water is a major challenge in forensic medicine. Diatom testing is considered a relatively reliable method for diagnosing drowning, especially for identifying the cause of death of decomposed bodies in water. It is considered the best method ("gold standard") and is widely used in forensic practice. The national public security industry standard "Technical Specification for Diatom Testing in Forensic Science: Microwave Digestion-Vacuum Filtration-Microscopy Method (GA / T1662-2019)" has been widely used in forensic practice. However, when this method is applied to diatom testing of drowned bodies in turbid water, the following problems exist:

[0003] Turbid water bodies usually contain a large number of suspended silica particles (such as mud and sand). These particles enter the lungs along with the drowning fluid during drowning. A small number of tiny silica particles (including diatoms) will further enter the liver, kidneys and other systemic organs through blood circulation. For systemic organs (such as the liver and kidneys), due to the small number of silica particles and high dispersion, it is easy to distinguish diatoms and other silica particles by morphological characteristics under a microscope. Therefore, they can be tested according to the GA / T1662-2019 method. However, for the lungs, samples obtained by microwave digestion and vacuum filtration often contain a large number of silica particles. Diatoms are covered or blocked by other silica particles, resulting in: (1) only a small number of diatoms can be detected under a microscope or no diatoms can be detected; (2) the morphological characteristics of diatoms are not obvious, making it difficult to accurately identify them, which is not conducive to the diagnosis of drowning and the inference of the drowning location.

[0004] In recent years, to address the interference of siliceous dust (caused by long-term inhalation from the air) in diatom analysis of lung tissues of middle-aged and elderly deceased, some scholars have adopted sodium polytungstate heavy liquid separation with a density of 1.8-2.4g / mL. However, when this method is directly applied to diatom analysis of lung tissues of drowned corpses in turbid water bodies, it has the following shortcomings:

[0005] (1) The above-mentioned sodium polytungstate heavy liquid is relatively expensive and has a wide density range. It requires continuous testing to determine the appropriate density of the heavy liquid, which is costly, time-consuming and labor-intensive.

[0006] (2) The siliceous impurity particles in the lungs of drowned corpses in turbid water bodies and the siliceous dust in the lung tissues of middle-aged and elderly deceased (due to long-term inhalation from the air before death) are very different in density, shape, size, composition, etc., so the above-mentioned heavy liquid separation method cannot be directly applied.

[0007] (3) The types, shapes, sizes, and densities of diatoms in different water bodies are different. For the precise extraction of diatoms in different water bodies, a heavy liquid that matches their density should be selected.

[0008] (4) In the past, the density of heavy liquid was often determined based on the range of diatom density reported in the literature. However, the diatom density reported in the literature is generally the density of undigested diatoms containing organic matter (i.e., the density of live diatoms). The samples after lung digestion contain inorganic diatom shells, and their density is very different from the former.

[0009] Therefore, for diatom testing of the lungs of drowned corpses in turbid water, the existing methods are difficult to accurately extract diatoms and need to be improved. Summary of the Invention

[0010] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for extracting diatoms in the lungs of drowned corpses in turbid water bodies. The present invention can effectively solve the problems of low diatom extraction rate and inaccurate diatom classification caused by interference of siliceous impurities when examining diatoms in the lungs of drowned corpses in turbid water bodies.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] A method for extracting diatoms from the lungs of drowned corpses in turbid water comprises the following steps:

[0013] (1) Determination of the skeleton density of diatoms and siliceous impurities in specific water bodies: a. Determination of diatom skeleton density: The dominant algae contained in the water sample from the suspected drowning site were cultured in the laboratory, freeze-dried after reaching the platform growth stage, thoroughly digested with nitric acid, and freeze-dried again. The diatom skeleton density ρ was measured using a gas displacement method true density meter. 硅藻 ;

[0014] b Determination of siliceous impurity skeleton density: Collect deep dry soil around the water sample at the suspected drowning site, remove plant roots and stems, and take 5.0-10.0g of it. Dissolve it thoroughly with nitric acid. After the digestion liquid is freeze-dried, the skeleton density of siliceous impurities ρ is measured using a gas displacement method true density meter. 硅质杂质 ;

[0015] (2) Preparation of heavy liquid:

[0016] According to the above-measured diatom skeleton density and siliceous impurity skeleton density, a heavy liquid with a density between the two is prepared;

[0017] (3) Microwave digestion of lung tissue:

[0018] About 2.0 g of lung tissue was taken and microwave digested according to the GA / T1662-2019 standard;

[0019] (4) Vacuum filtration (I):

[0020] The liquid obtained by digestion in step (3) is vacuum filtered to allow particulate matter such as diatom shells to adhere to the filter membrane. The microporous filter membrane used is a polyester nuclear track etched filter membrane (PETE) with a pore size of 1-3 μm. The membrane is a nuclear pore membrane with a pore structure, which can effectively enrich diatoms. Compared with commonly used filter membranes made of materials such as acetate fiber, nylon, and polyethersulfone, which are formed by interlacing multiple layers of fibers, the membrane has better permeability of tiny impurity particles and fewer impurities on the filter membrane.

[0021] (5) Ultrasonic desorption:

[0022] The filter membrane obtained by filtration in step (4) is subjected to ultrasonic desorption using the heavy liquid prepared in step (2).

[0023] (6) Centrifugation

[0024] Centrifuging the liquid obtained in step (5) and retaining the supernatant and the precipitate;

[0025] (7) Diatom heavy liquid extraction:

[0026] Add the heavy liquid prepared in step (2) to the precipitate in step (6), shake and centrifuge, and collect the supernatant;

[0027] (8) Vacuum filtration (II):

[0028] The supernatants collected in steps (6) and (7) were combined and filtered using the same microporous membrane as that used in vacuum filtration (I), namely a polyester core track etched membrane (PETE) with a pore size of 1-3 μm.

[0029] (9) Coating:

[0030] A gold film or a platinum film is plated on the surface of the filter membrane obtained in step (8) using a vacuum coating device, and the thickness is controlled to be 5-10 nm.

[0031] The filter membrane sample obtained in step (9) was subjected to scanning electron microscopy according to the GA / T1662-2019 standard.

[0032] Preferably, the preparation method of the heavy liquid in step (2) is as follows: zinc bromide is used as the solute, 0.5% to 1.0% sodium 6-metaphosphate solution is prepared as the solvent, and the solute is added so that the density of the prepared heavy liquid is ρ 重液 In ρ 硅藻 -ρ 硅质杂质 The addition of sodium 6-metaphosphate can reduce surface tension, maintain the stability of the dispersion system, and prevent particle aggregation. Compared with the prior art, the present invention has the following innovations and beneficial effects:

[0033] 1. For the first time, the skeleton density of diatoms (density of non-living diatoms) and the skeleton density of siliceous impurities contained in water samples from suspected drowning sites were obtained through digestion, freeze-drying, gas replacement method and true density meter measurement. On this basis, the density of the heavy liquid used to separate inorganic diatom shells was determined. The process is precise and scientific.

[0034] 2. The cost of the sodium polytungstate heavy liquid currently used to extract diatoms is too high. The new heavy liquid formula uses zinc bromide as the main solute, which significantly reduces the cost.

[0035] 3. For the first time, the dispersant sodium 6-metaphosphate was added to the heavy liquid formula to enhance the stability of the heavy liquid suspension system, prevent the diatom shells from agglomerating and settling with impurity particles, and improve the diatom recovery rate.

[0036] 4. For the first time, a nuclear pore membrane with a pore size of 1-3 μm (pore structure, such as Figure 5 ) replaces the filter membranes (multi-layer fiber interlaced structure) with a pore size of 0.45μm made of acetate fiber, nylon, polyethersulfone and other materials currently widely used in forensic diatom testing. It has a large filtering capacity for tiny siliceous impurities, less impurity interference and higher detection accuracy.

[0037] 5. The present invention has significant innovations in the diatom extraction process:

[0038] The existing diatom testing standard GA / T1662-2019 specifies that the extraction process for diatoms from drowned lungs includes microwave digestion, vacuum filtration, and film coating. This invention addresses the problems of low diatom extraction efficiency, significant impurity interference, and large testing errors in existing methods for testing lung tissue from drowned bodies in turbid water. The method uses a nine-step process: determination of the skeleton density of diatoms and siliceous impurities contained in specific water, preparation of a heavy liquid, microwave digestion of lung tissue, vacuum filtration (I), ultrasonic desorption, centrifugation, diatom heavy liquid extraction, vacuum filtration (II), and film coating. These steps are closely linked and essential.

[0039] In summary, compared with the existing technology, the present invention has great innovation in the pretreatment of lung tissue samples with many impurities. It has the advantages of accurate selection of heavy liquid density, high diatom extraction rate, less impurity interference, and accurate results. It is of great significance to improve the value of diatom testing in drowning diagnosis and drowning location inference, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a comparison chart of the effects of the nuclear pore membrane of the present invention and the membrane used in the current standard;

[0041] Figure 2 This is a comparison chart of the effects before and after using the dispersant;

[0042] Figure 3The left picture shows the electron microscopic observation effect of the current standard, and the right picture shows the electron microscopic observation effect of the present invention;

[0043] Figure 4 The left picture shows the effect of electron microscopy of the current standard, and the right picture shows the effect of electron microscopy of the present invention;

[0044] Figure 5 This is a scanning electron microscope image of the polyester nuclear track etched filter membrane used in the present invention;

[0045] Figure 6 This is a scanning electron microscope image of polyethersulfone microporous filter membrane;

[0046] Figure 7 This is a scanning electron microscope image of the polytetrafluoroethylene filter membrane. DETAILED DESCRIPTION

[0047] Example 1: Extraction of diatoms from the lungs of drowned corpses in turbid water

[0048] 1. Determination of the skeleton density of diatoms and siliceous impurities in turbid water samples from suspected drowning sites (1) Determination of the skeleton density of diatoms

[0049] The dominant algae, Nitzschia, contained in the water sample from the suspected drowning site was cultured in the laboratory, freeze-dried after reaching the platform growth stage, thoroughly digested with nitric acid, and freeze-dried again. The diatom skeleton density ρ was measured using a gas displacement true density meter. 硅藻 1.5532 g / cm 3 .

[0050] (2) Determination of siliceous impurity skeleton density

[0051] Deep dry soil around the suspected drowning site was collected. After removing plant roots and stems, 10.0 g of the soil was thoroughly digested with nitric acid. The liquid was freeze-dried and 3.5 cc of soil powder was taken. The skeleton density of the siliceous impurities (ρ) was measured using a gas displacement true density meter. 硅质杂质 2.6120g / cm 3 .

[0052] 2. Preparation of heavy liquid

[0053] Prepare a heavy liquid with a density between the skeleton density of diatoms contained in the on-site water and the skeleton density of siliceous impurities. The preparation method is as follows:

[0054] Prepare a heavy liquid with zinc bromide powder as solute and pure water as solvent, add dispersant sodium 6-metaphosphate to a concentration of 1.0%, and adjust the amount of zinc bromide to make the density of the heavy liquid 2.3200 g / cm 3 .

[0055] 3. Microwave digestion of lung tissue

[0056] About 2.0 g of lung tissue was taken and microwave digested according to the GA / T1662-2019 standard.

[0057] 4. Vacuum filtration (I)

[0058] The digested liquid is vacuum filtered using a polyester nuclear track etched filter membrane with a pore size of 3 μm, allowing particles such as diatom shells to adhere to the filter membrane. The microporous filter membrane used is a polyester nuclear track etched filter membrane (PETE) with a pore size of 1-3 μm. This membrane is a nuclear pore membrane with a porous structure that can effectively enrich diatoms. Compared with filter membranes made of materials such as acetate fiber, nylon, and polyethersulfone, which are formed by interlacing multiple layers of fibers, it has better permeability for tiny impurity particles and fewer impurities on the filter membrane.

[0059] 5. Ultrasonic desorption

[0060] Place the filter membrane obtained by filtration into a centrifuge tube, add the prepared heavy liquid to completely submerge the membrane, place it in 400W ultrasound for desorption for 150s, let it stand for 60 minutes and then centrifuge, retaining the supernatant and sediment separately.

[0061] 6. Centrifugal

[0062] The liquid obtained in step (5) is centrifuged, and the supernatant and the precipitate are retained.

[0063] 7. Diatom heavy liquid extraction

[0064] The prepared heavy liquid was added to the sediment, shaken, allowed to stand for 60 minutes, and then centrifuged to collect the supernatant.

[0065] 8. Vacuum filtration (II)

[0066] The supernatants collected in steps 6 and 7 were combined and filtered using the same microporous membrane as used in vacuum filtration (I), namely a polyester core track etched membrane (PETE) with a pore size of 1-3 μm.

[0067] 9. Coating

[0068] A gold film with a thickness of 10 nm was deposited on the surface of the filter membrane using vacuum coating equipment.

[0069] Afterwards, the prepared filter membrane samples were subjected to scanning electron microscopy testing in accordance with the GA / T1662-2019 standard.

[0070] Comparison of test results

[0071] 1. Comparison between nuclear pore membrane and membranes used in current standards or conventional methods

[0072] Figure 1 The left picture shows polyethersulfone microporous membrane. Figure 1The right picture shows a polyester nuclear track etched filter membrane (the membrane used in the present invention), and the pore size of both membranes is 3 μm. Figure 1 The left picture shows that the polyethersulfone microporous filter membrane has a multi-layer fiber interlaced structure, and the right picture shows that it has a pore structure. This pore structure can significantly increase the filtration capacity of tiny siliceous impurities, reduce impurity interference, and improve detection accuracy.

[0073] 2. Comparison of effects before and after use of dispersant

[0074] Experimental studies have shown that the use of dispersants can prevent particle agglomeration, improve the recovery rate of diatoms, and filter out some tiny siliceous particles, making the background clearer (see Figure 2 ).

[0075] 3. Comparison of diatom recovery rates between the present invention and current standards

[0076] Lung tissue from people who died on land and were confirmed to be free of diatoms was used as a blank sample for the addition experiment. 2 g of lung tissue was digested with microwaves, and then 1 mL of a diatom shell suspension with a concentration of 18245 cells / mL and 0.02 g of liquid from wet soil digestion were added to the digestion solution. The samples were then processed according to the current standards (including vacuum filtration and coating) and the present invention (including vacuum filtration (I), ultrasonic desorption, heavy liquid extraction, vacuum filtration (II), and coating), and finally detected using a scanning electron microscope.

[0077] The results show that the recovery rate of diatoms of the present invention is 83.9±1.4%, while the recovery rate of diatoms of the current standard is less than 10%. This is because: when the current standard is used for detection, most of the diatoms are covered by impurities such as mud and sand, while the present invention can effectively remove impurities (see Figure 3 ).

[0078] 4. Comparison of the application effects of this invention and the current standard

[0079] The lung tissue of a drowned corpse in the turbid water was analyzed according to the current standard (GA / T1662-2019 method) and Example 1 of the present invention. The results are as follows: Figure 4 , as shown in Table 1:

[0080] Figure 4 The left picture shows the observation effect of the current standard electron microscope, and the right picture shows the observation effect of the electron microscope of the present invention. Figure 4 It can be seen that according to the current standard test, the filter membrane is covered by impurities, the filter membrane holes are not visible, and the diatoms are covered by impurities, so the detection rate is low; in contrast, according to the present invention, there is less interference from impurities, the filter membrane holes are clearly visible, and the diatom detection rate is high.

[0081] According to literature, a lung-to-water ratio (L / D) greater than 2 strongly indicates drowning; a value less than 2 suggests both drowning and non-drowning, and the cause of death must be diagnosed based on other evidence. As shown in Table 1, the present invention's L / D ratio is 50.1, strongly indicative of drowning. This demonstrates that the present invention overcomes interference from impurities in lung tissue samples, resulting in higher detection sensitivity and better drowning diagnosis than existing methods.

[0082] Furthermore, as can be seen from Table 1, compared with the current standards, the types of diatoms in the lungs and in the water are more consistent when tested using the present invention, and the results are more indicative of the drowning location.

[0083] Therefore, the present invention has greater application value in drowning diagnosis and drowning location inference.

[0084] Table 1 Comparison of diatom test results of lung tissue of drowned corpses

[0085]

Claims

1. A method for extracting diatoms from the lungs of drowned corpses in turbid water, characterized in that The steps include: (1) Determination of the skeleton density of diatoms and siliceous impurities contained in specific water bodies: a Determination of diatom skeleton density: The dominant algae contained in the water sample from the suspected drowning site were cultured in the laboratory, freeze-dried after reaching the platform growth stage, thoroughly digested with nitric acid, and freeze-dried again. The diatom skeleton density ρ was measured using a gas displacement method true density meter. 硅藻 ; b Determination of siliceous impurity skeleton density: Collect deep dry soil around the water sample at the suspected drowning site, remove plant roots and stems, and take 5.0-10.0g of it. Dissolve it thoroughly with nitric acid. After the digestion liquid is freeze-dried, the skeleton density of siliceous impurities ρ is measured using a gas displacement method true density meter. 硅质杂质 ; (2) Preparation of heavy liquid: According to the above-measured diatom skeleton density and siliceous impurity skeleton density, a heavy liquid with a density between the two is prepared; (3) Microwave digestion of lung tissue: About 2.0 g of lung tissue was taken and microwave digested according to the GA / T1662-2019 standard; (4) Vacuum filtration (I): The liquid obtained by the digestion in step (3) is vacuum filtered to allow the particulate matter to adhere to the filter membrane, and the microporous filter membrane used is a polyester core track etched filter membrane with a pore size of 1-3 μm; (5) Ultrasonic desorption: The filter membrane obtained by filtration in step (4) is subjected to ultrasonic desorption using the heavy liquid prepared in step (2); (6) Centrifugation Centrifuging the liquid obtained in step (5) and retaining the supernatant and the precipitate; (7) Diatom heavy liquid extraction: Add the heavy liquid prepared in step (2) to the precipitate, shake and centrifuge, and collect the supernatant; (8) Vacuum filtration (II): The supernatants collected in steps (6) and (7) were combined and filtered using the same microporous filter membrane as used in vacuum filtration (I), i.e., a polyester core track etched filter membrane with a pore size of 1-3 μm; (9) Coating treatment: The surface of the filter membrane obtained in step (8) is coated with a gold film or a platinum film with a thickness of 5-10 nm using a vacuum coating device.

2. The method for extracting diatoms from the lungs of drowned corpses in turbid water as claimed in claim 1, characterized in that The preparation method of the heavy liquid in step (2) is as follows: Use zinc bromide as solute and 0.5% to 1.0% sodium 6-metaphosphate solution as solvent. Add solute so that the density of the prepared heavy liquid is ρ 重液 In ρ 硅藻 -ρ 硅质杂质 within the range.

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