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Application of a simple enzymatic digestion method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis

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Application of a simple enzymatic digestion method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis

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Abstract

The reliability and applicability of quantitative and qualitative diatom analysis by an enzymatic digestion method in the diagnosis of drowning of putrified bodies has been evaluated. The authors report the analysis of water and organ samples of 12 immersion cases using light microscopy. This study included control organ samples from the bodies of persons who died from causes other than drowning. Organ samples were treated by both chemical and enzymatic methods, the first one using concentrated nitric acid and the second proteinase K. Diatoms were present in most organ samples of the immersed corpses; no diatoms could be found in the control samples. Our experience was that the enzymatic method seemed to be more convenient in terms of rapidity, safety and environmental protection than chemical digestion. The number of diatoms recovered with both methods was similar. Qualitative and quantitative analysis of both water and organ samples of immersion cases supported the diagnosis of death by drowning in 41.6% of the putrified cases studied. The authors suggest that diatom analysis using enzymatic digestion of organs can be used as a criterion for positive diagnosis of drowning in cases involving putrified bodies.

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... Ludes et al designed this method of enzymatic digestion for the diagnosis of putrefied bodies. Tissues are treated with both chemical and enzymatic reagents: buffer Tris-HCl (pH 7.5) and proteinase K [49]. containing 2% SDS. ...
... The mixture was incubated at 50°C overnight, 500 ml proteinase K was added, and the solution was diluted with 100 ml of distilled water and centrifuged at 3000 rpm for 15 min, after which the upper layer was removed. The sediment was transferred to a slide and examined under the light microscope [49]. ...
... A common recommendation in the reviewed works is that all of the materials (gloves, filters, etc.) and reagents must be carefully treated to avoid contamination. Other materials (surgical instruments, containers, tubes, etc.) should be washed with double distilled water [28,49,61] or deionized water and dried in an autoclave [43]. ...
Chapter
Diatoms are single-celled organisms widely distributed in aquatic environments with several applications, such as oil exploration, environmental indications, microbial ecology, or forensic examinations. In this chapter, we analyse forensic applications of diatom analysis, including the usefulness of the test in the diagnosis of drowning, identification of the drowning site, identification of the suspect and information about the time of death. We also address the sample preparation process and review the most used techniques for diatom analysis involving the digestion of tissues from specific organs to study (acid digestion, acid digestion in disorganization can, Soluene-350 digestion, enzymatic digestion, membrane filter methods, and some novel techniques). Finally, we review some protocols in diatom analysis and histological findings in drowning.
... In order to solve this problem, some forensic experts suggested the use of quantification of the measurement of diatoms in the lung tissues. Ludes et al 9 have suggested a cut-off concentration of 20 diatoms per 10 g of the lung tissue. However, the concentration of diatoms in the drowning medium varies greatly. ...
... Diatom tests of the closed organs, such as liver, kidneys or bone marrow, are often negative in the drowning cases. 4,9,18 If there are no diatoms detected in the liver, kidneys or bone marrow of the drowning cases, we may have no confidence in determining whether the suspicious death is due to drowning. For example, a 25 years old victim was found in a ditch. ...
Article
The presence of diatoms in the lung tissues, internal organs and bone marrow is considered as the supportive evidence in the diagnosis of death by drowning. Generally, the diatoms detected in the lung tissues are regarded as insignificant since these diatoms can be detected in the lung tissues of the postmortem immersion bodies. In this study, we analyzed the relationships between the numbers of the diatoms in the lung tissues and the drowning medium. We made a comparison analysis between the diatoms in the lung tissues and the drowning medium using the ratio of diatom numbers in both samples (L/D ratio), utilizing Microwave Digestion - Vacuum Filtration - Automated Scanning Electron Microscopy method. Our data indicate that the L/D ratios in victims of the drowning group were higher than the postmortem immersion group. A higher L/D ratio provides valuable information about the cause of death in drowning victims. Quantitative diatom analysis in the lung tissues, especially combined with the diatom analysis of the drowning medium, provides supportive evidence in determining if a body recovered in water was due to drowning or not.
... Diatom test gains importance for diagnosis and confirmation of drowning in such cases as due to their hard silicaceous skeleton, they can be recovered from putrefied or burnt tissues by either enzymatic or acid digestion [4]. Diatoms are unicellular plant that has most distinctive features of crystalline extracellular coat or frustules composed of silica and having unique pattern of symmetry and microstructure. ...
... Seasonal distribution pattern of cases showed that the diatom positive cases were more in the rainy (13) & summer season (8) as compared to spring (4) and winter (3). The gender distribution clearly deviated towards males with 40 males & 10 females. ...
Article
Full-text available
Presently, forensic pathologists all over the world are divided over reliability of diatom test as a diagnostic aid. The aim of the present study was to test the reliability of diatom test as indicator of antemortem drowning by comparing the presence of diatoms in dead bodies found in water, in cases where suspected cause was drowning with those cases where another cause of death was established. Specimens were preserved for diatom test at the time of postmortem examination. These specimens were subjected to acid digestion for extraction of bone marrow to conduct diatom test. Water sample was collected from the site of recovery of the bodies. The specimens were subjected to diatom test for detection of diatoms and comparison of types of diatoms found in water & bone sample respectively. Amongst the 41 cases, 28 cases showed positive results and 13 cases showed negative results for diatom tests. Seasonal distribution pattern of cases showed that the diatom positive cases were more in the rainy (13) & summer season (8) as compared to spring (4) and winter (3). The gender distribution clearly deviated towards males with 40 males & 10 females. In 11 cases where other cause of death except drowning was established at the postmortem examination, diatom test were negative in all the cases. In cases where other findings of drowning are lost to decomposition, detection of identical diatoms in body and putative drowning medium should be considered as corner stone for establishing ante-mortem drowning.
... The diatom test is widely regarded as one of the most reliable evidence for diagnosing death by drowning (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). During the last decades, a lot of forensic diatom test methods have been introduced in forensic sciences (5,6,10,12,13). ...
... The diatom test is widely regarded as one of the most reliable evidence for diagnosing death by drowning (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). During the last decades, a lot of forensic diatom test methods have been introduced in forensic sciences (5,6,10,12,13). The silica-based frustules of diatoms are detected in the lung, liver, kidney, and bone marrow. In those methods, false-negative results are easily obtained because lots of diatoms are lost during centrifugation process. ...
Article
Diagnosis of drowning remains a difficult issue in current forensic sciences. A large number of diatoms were lost by removing the supernatant after centrifugation in the conventional forensic diatom test. We developed a novel membrane filtration method to enrich diatoms from samples. A new solution using different ratios of acetic acid and eugenol is prepared to make the membrane transparent. These processes allow the diatom-containing membrane to be visualized and identified easily by light microscopy. The tissues contaminated by water rich in diatoms were detected by the new method for the recovery of diatoms. Eleven drowning cases were analyzed by both the new method and the conventional method to compare the sensitivity of both methods. The recovery of the novel diatom test method was 54.2 ± 23.1%. The positive rate of the novel method has been proven to be superior to the conventional method in the diagnosis of drowning.
... Thus diatoms can be used as an important tool in the diagnosis of drowning cases. Various workers supported the reliability of the diatom test (Ludes et al., 1994;Pollanen, 1998;Vinayak et al., 2010;Verma, 2013) [17,18] . Diatom test is frequently used in forensic science laboratories in India indrowning diagnosis, but at the same time, a large pool of forensic experts is confused about the practicality and reliability of the test for ante-mortem drowning detection. ...
... Thus diatoms can be used as an important tool in the diagnosis of drowning cases. Various workers supported the reliability of the diatom test (Ludes et al., 1994;Pollanen, 1998;Vinayak et al., 2010;Verma, 2013) [17,18] . Diatom test is frequently used in forensic science laboratories in India indrowning diagnosis, but at the same time, a large pool of forensic experts is confused about the practicality and reliability of the test for ante-mortem drowning detection. ...
... Timperman (1972) emphasised that the presence of even a single diatom in femoral bone marrow is consistent with drowning as a cause of death. However, diatom test should be considered positive when number of diatom is found at least 20/100µl of pellet obtained from 10 g lung sample and 5 from other organs (Ludes et al., 1994). Lucas et al. (2002) studied 123 dead bodies, in these 97 bodies were pertaining to drowning and intake of ethanol and illicit drugs was found to be the contributory intoxicants in 53% cases of accidental drowning. ...
... In our study, the presence of diatoms is ~50 per 50 g sternum marrow which is far above the minimal established limit, i.e. 5 complete diatoms/10 g tissue except of lung by Ludes et al. (1994). Control water sample also contain significant number of diatoms. ...
Article
Full-text available
The aim of this study was to examine the combined contribution of diatom and toxicological tests to establish the cause of death of a body found in a swimming pool of a farmhouse. The presence of abrasion on the forehead of the dead body in police investigation was considered that this might be a case of murder. For the diagnosis of suspected drowning, diatom profiles of sternum and control water sample were made and carefully evaluated. The diatom study in sternum revealed that there was presence of numerous diatom frustules in the marrow extract which were similar to the diatoms of control water sample. It clearly indicates that the sternal diatoms originated from the pool water. Therefore, diatom test was found to be helpful in proving the cause and site of death and in excluding the possibility of post mortem drowning. Surprisingly toxicological findings in the viscera and blood sample of the deceased gave positive tests for the presence of morphine alkaloid and ethyl alcohol (103.50 mg/100 ml in the blood). The circumstantial evidence at the farmhouse were indicative of a rave party in which liquor was served and probably the party goers were habitual of using drugs to enjoy the taste of swimming under the influence of intoxication. Therefore, on the basis of findings of toxicological examination, possibility of accidental drowning could not be ruled out.
... This method can be used where there are limitations in budget, equipment, training (Kakizaki et al., 2019). To eliminate the effects of the use of concentrated acids, they can be used to prepare proteinase K drugs (Ludes, Quantin, Coste, Mangin, 1994). The authors proved the same effectiveness of this technique as when using concentrated sulfuric and nitric acids. ...
Article
Full-text available
The article describes the current possibilities of forensic medicine in the diagnosis of death by drowning, as well as reflects the different views of the authors and the evolution of approaches to this issue. Despite the constant interest of scientists in the topic of drowning and extensive research on this issue, there are still many white spots. Researchers are trying to identify more specific changes characteristic of drowning and possible methods to detect them. The main evidence based method of diagnosing drowning at present is the detection of diatomic plankton in the tissues of isolated kidneys by light optical microscopy. However, microscopic examination and identification of diatomic plankton require a lot of time and accurate taxonomic examination. Also, a disadvantage of this method is that the methods of processing the material sent for research, as a result of the use of concentrated acids for the destruction of organs, greatly complicates and sometimes eliminates the possibility of detection of diatomic plankton. In some cases, the presence of diatoms during drowning is so small that it is impossible to detect them by microscopy. In such cases, the presence of phytoplankton DNA detected by PCR in tissues during drowning is almost the only method for diagnosing drowning. Nowadays there has been a lack of research in the field of forensic medicine related to the use of evidence-based medicine, especially in the field of drowning.
... The authors had no possibility of investigating other potential body parts where diatoms might be found, e.g. femoral bone marrow [16] or brain tissue [19]. ...
Article
Full-text available
The study addresses the utility of diatoms in deciding whether a woman whose body was found in a river had died as a result of drowning. Several hours after the woman had been reported missing, a rescue dog led the police to a concrete water tank close to her workplace. Her body was found two months later in a nearby river. The composition and abundance of diatoms found in the victim’s internal organs were compared to those in water samples from the location where the corpse was found and from other potential sites of drowning. These locations proved to support quite different assemblages of diatoms. The clumps of diatoms encountered in the lung proved useful for interpreting the place of drowning. The results suggest that death occurred as a result of drowning in the tank and the body was subsequently moved to the river, thereby facilitating secondary diatom introductions.
... [9] reported in 50% of cases. Caniero, et al [39], Bak, et al [51], Benzamin, et al. [38], Kakizaki [52], Yoshimura, et al. [53], Ludes, et al. [55] and Kakizaki and Yukawa [56] reported positivity of diatoms in 100% of cases. The negativity of diatoms in drowning deaths might be due to low abundance of diatoms in drowning medium, destruction or loss of diatoms during processing or inappropriate organ samples used for the study. ...
... Plankton test is mainly used for diagnosis of drowning; however, seawater may enter various organs while floating. In the case of a long interval after death, determination of cause of death and estimation of time since death are more difficult [19][20][21]. Many of these techniques are inapplicable to a skeletonized body, in particular. ...
Article
Full-text available
The present investigation was performed with the objective of developing a method to estimate how long a corpse had been immersed in water after death (the time since death). Accurate determination of the time elapsed since death may lead to identification of the place of drowning, and therefore, serves not only as a piece of information useful for determination of the cause of death but also leads to prompt identification of the body. The results showed that diatoms attached to the surface of dental enamel increased with prolongation of immersion time in water. Further, as the immersion time increased, the quantity of O, Si, Mg, K, Al, and S detected on the surface of dental enamel increased, while the quantity of the main dental components (Ca and P) that were detected gradually decreased. Based on these results, we calculated a regression formula to estimate the immersion time. Our method is considered to be a breakthrough technique for evaluating the time since death more objectively, compared to the conventional method of determination based on the degree of decomposition of the corpse.
... Thus diatoms can be used as an important tool in the diagnosis of drowning cases. Various workers supported the reliability of the diatom test (Ludes et al., 1994;Vinayak et al., 2010;Verma, 2013). Diatom test is frequently used in forensic science laboratories in India in drowning diagnosis, but at the same time, a large pool of forensic experts is confused about the practicality and reliability of the test for ante-mortem drowning detection. ...
Article
Full-text available
In the present report, 38 human drowning case studies from Haryana (India) were examined and solved by using “diatom test” in Forensic Science Laboratory Madhuban, Karnal (Haryana). After medico-legal examination, crime exhibits were sent to the laboratory along with the samples of water from which the dead bodies were recovered. Exhibits were treated with acid digestion, centrifugation and later on analyzed under the microscope for the presence of diatoms if any. Out of 38 cases, 34 cases were found to be positive for the diatom test as the crime exhibits (sternum, clavicle, liver, lungs, spleen, kidney, intestine, heart and putrefied viscera) had the same diatom types as were present in water samples from the drowning sites of the respective cases.
... Diatom frustules can be easily extracted from internal organs of a drowning victim, usually whole and intact, sometimes fragmented but still recognizable [30], just because all diatoms have a siliceous skeleton frustule (made up of two valves fitted together by a connective tissue) resistant to putrefaction and degradation via enzymatic and acid digestion [42,45,46]. Most of the diatom sample treatment is based on the digestion of tissue samples by strong acid because it is cheap and known to be a reliable technique [24-27, 34, 42, 45-47]. ...
Article
Full-text available
In this preliminary study, a digestive method used in forensic context to extract diatoms has been applied in organs taken from ten wildlife animals belonging three species of mammals (a deer, a roe and five otters) and one species of birds (two magpies). Only four carcasses were recovered from aquatic environments (bath trough for animals, bathtub irrigation, river) and only in three cases out of ten that the cause of death was ruled out as drowning. In seven cases, the death was due to other causes: gunshot injuries for one otter, blunt trauma for a magpie, and traumatic injuries followed by motor vehicle collision in other four otters and a roe. Post-mortem examination was performed in all carcasses. The diatom test protocol was performed according to the Italian guidelines for analysis of benthic diatoms for ecological status assessment of inland waters. Five grams of lung, liver, and kidney was taken from all the animal carcasses. In some cases, additional tissue samples were also available among which brain, heart, spleen, and bone marrow. In all four cases found in water, the drowning medium was also available. Hydrogen peroxide (H2O2) digestion was performed, and identification of 21 genera of diatoms was obtained. The method proved to be suitable for the identification of diatoms in the organs of the animals drowned supporting the final diagnosis of drowning. Only in otters, all died for causes other than drowning, diatoms did not prove to be suitable for the diagnosis of drowning since their presence in the internal organs was mainly related to their main diet based on fishmeal. The authors believe that this first trial is very promising, and the results suggest that diatom test can be easily applied in forensic veterinary context.
... Thus diatoms can be used as an important tool in the diagnosis of drowning cases. Various workers supported the reliability of the diatom test (Ludes et al., 1994;Vinayak et al., 2010;Verma, 2013). Diatom test is frequently used in forensic science laboratories in India in drowning diagnosis, but at the same time, a large pool of forensic experts is confused about the practicality and reliability of the test for ante-mortem drowning detection. ...
Article
Full-text available
In the present report, 38 human drowning case studies from Haryana (India) were examined and solved by using “diatom test” in Forensic Science Laboratory Madhuban, Karnal (Haryana). After medico-legal examination, crime exhibits were sent to the laboratory along with the samples of water from which the dead bodies were recovered. Exhibits were treated with acid digestion, centrifugation and later on analyzed under the microscope for the presence of diatoms if any. Out of 38 cases, 34 cases were found to be positive for the diatom test as the crime exhibits (sternum, clavicle, liver, lungs, spleen, kidney, intestine, heart and putrefied viscera) had the same diatom types as were present in water samples from the drowning sites of the respective cases.
... According to Farrugia & Ludes (2011), the diatom test can be considered positive when at least 20 diatom valves are identified per 100 mL of sediment extracted from a 2 g lung sample, and identification of more than 5 complete diatom valves per 100 mL of sediment extracted from a 2 g tissue samples such as brain, kidney, liver and bone marrow. The same values were suggested by both Ludes et al. (1994) and Bortolotti et al. (2011). In some case studies, however, the presence of low numbers of valves from cosmopolitan diatoms in bone marrow (1-2) have been considered positive. ...
Article
Full-text available
Diatoms are unicellular, photosynthetic, eukaryotic organisms often classified as among the algae. There are around 15 000 known species, but many more have yet to be described. Their uniqueness lies in the siliceous covering of the cell, each being encased in a pair of silica valves. Silica is virtually inert and indestructible, so after the organism’s death the silica parts remain. The silica parts provide information for the classification of these diverse organisms. Diatoms have been used in forensic science in a variety of ways, the most frequent being the diagnosis of death by drowning. When a person drowns, water will enter the lungs and then enter the bloodstream through ruptures in the peripheral alveoli before being carried to the other organs such as the liver and heart. Naturally, the microscopic contents of the water, which will include diatoms, will pass into the blood as well. The detection of diatoms in the organs can contribute to a diagnosis of death by drowning, a process referred to as the ‘diatom test’. We will discuss this test in more detail, illustrating our discussion with real examples.
... The analysis of the frequency of detection of diatoms in bodies of people who died of drowning indicates the tendency of decreasing the frequency of their determination in recent years. Such a phenomenon may be due to the method of destruction of the material destined for research, as a result of the use of concentrated acids for the destruction of organs, which sometimes eliminates the possibility of detecting diatomaceous plankton [19,[36][37]. Therefore, the PCR test method proved to be more reliable, sensitive, specific and rapid compared with the diagnosis of the usual diatoms by acid digestion. ...
Article
Full-text available
Forensic medical diagnostics of drowning now is a difficult issue to resolve. Determination of diatom plankton with light microscopy is one of the supplementary methods for diagnostics of drowning. The disadvantage of this method is the use of concentrated acids to destroy the tissues of the organs, which greatly complicates, and sometimes precludes the detection of diatom plankton. In this case, the detection of other phytoplankton species in internal organs is treated as pseudoplankton, but does not have a diagnostic value. We have developed a sensitive and specific method of drowning diagnostics using a pair of specific oligonucleotide primers by the polymerase chain reaction (PCR) method to determine the presence of DNA of Cyanobacteria of the genus Microcystis, namely a fragment of the 16S rRNA gene in the tissues of mice and water samples in order to establish the fact and place of drowning. In order to evaluate the diagnostic value of this method, we conducted an experimental study to detect fragments of the 16S rRNA gene in mice tissues during drowning and post-mortem immersion. The amplification products were found in the tissues of heart, kidneys, liver, spleen, bone tissue, brain tissue, and lungs in case of drowning. During post-mortem immersion products of amplification are detected only in the tissues of lungs. The results indicate that the proposed PCR method is a potentially useful tool for diagnosing of mechanical asphyxia as a result of drowning.
... There are diatoms found in the bodies died from drowning and causes other than drowning, which led to a strong divergence of views concerning the value of the diagnostic significance of diatom test (2)(3)(4)(5)(6)(7)(8)(9). The conventional diatom test consists of digestion of organic matter, centrifugation, and observation by light microscopy (3,4,(10)(11)(12)(13)(14). According to our previous study (15), the measured loss ratios of centrifugation ranged from 29.94% to 49.60% (34.18% ...
Article
Full-text available
The value of diatom test for the diagnosis of drowning remains controversial. The conventional forensic diatom test with low sensitivity is not a useful tool to provide accurate information about diatom in the tissues and organs. To improve the sensitivity of the diatom test, we developed a novel method called the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method which resulted in a high recovery of diatoms. In this article, we analyzed 128 water-related death cases. Our results showed that the MD-VF-Auto SEM method could achieve a much higher positive rate (0.97) in drowning cases. Large amounts of diatoms are retained, even concentrated, in the lung tissues during the intense breathing movement in drowning process. This might be useful for the diagnosis of drowning. Our research indicates that the MD-VF-Auto SEM method would be a valuable methodology in the study of diatom test for the forensic community.
... Diatom test has come up as a direct screening test to diagnose the deaths due to drowning. Diatoms detected in the drowned bodies have been deemed to be the most reliable indicator of drowning particularly in the absence of other evidentiary clues, (Hurlimann et al., 200;Kobayashi et al., 1993;Ludes et al., 1994;Ludes et al., 1996;Pollanen, 1996;Peabody, 1977 and1980;and Timperman, 1969and Timperman et al., 1972. ...
Article
Full-text available
ARTICLE INFO ABSTRACT Diatom frustules continue examined commonly during autopsies of deaths due to drowning. Diatoms are unicellular microorganisms which are commonly found in almost all water bodies. Their silica wall plays significant tool in forensic diatomology. Diatom analysis has been suggested to provide supportive evidence of drowning but the consistency and applicability of quantitative and qualitative diatom analysis in the diagnosis of drowning is still tentative in the literature. Diatom test has been extensively applied to detect post mortem or antemortem drowning and comparing the diatoms found in biological sample with those found in water sample indorses that death took place, probably in same water medium. Death by drowning is the result of encumbering of respiration by comprehensive or partial submersion and subsequent entry of water into the air passages. 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, lungs and bone marrow by circulation. Diatoms found inside the body of a drowned victim may serve as corroborative evidence in the diagnosis of cause of death. The diatom test stands as the only direct screening test for drowning. Copyright©2016, Dr. Rajeev Kumar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
... Until now, a lot of diatom test methods have been developed for the diagnosis of drowning since the first description of diatoms in drowning. The most common extraction technique consists of chemical digestion by nitric or sulfuric acid [6], soluene-350 [7,8], or enzymes [9,10]. The conventional acid digestion method consist of acid digestion, centrifugation, and observation by light microscopy. ...
Article
Objective: To evaluate the practical value of microwave digestion technique and scanning electron microscopy (SEM) for diatom test in diagnosis of drowning. Method: 105 autopsied cases were divided into three groups; drowning group (70 cases), post-mortem immersion group (15 cases who were thrown into water after death on land) and control group (20 cases died of natural causes on land). Qualitative and quantitative diatom tests were performed with both methods microwave digestion technique with SEM (method A) and nitric acid digestion with light microscopy (method B) in the lung, liver, kidney and bone marrow of them and in the water samples of drowning group. Results: The diatom-detectable rates in the drowning group were 100%, 94.3%, 92.9%, 82.9%, and 100% in lung, liver, kidney, bone marrow and water respectively with the method A; and 90%, 62.9%, 51.4% 28.6%, and 92.9%, with the method B. The positive rates of diatom test were 100% and 65.7% for method A and method B respectively. The types of diatom in organs of drowning group detected by both method A and method B were the same with the samples of water. A few diatoms (<3/2g) were also found in the lungs in the post-mortem immersion group with the two methods, but no diatom was detected in other organs of the post-mortem immersion group and in all organs of control group died of natural causes on land. Conclusion: Comparing with the method of nitric acid digestion with light microscopy, the method of microwave digestion technique with SEM is higher rate of detection, more sensitive and accurate to diatom test.
... [9] reported in 50% of cases. Caniero, et al [39], Bak, et al [51], Benzamin, et al. [38], Kakizaki [52], Yoshimura, et al. [53], Ludes, et al. [55] and Kakizaki and Yukawa [56] reported positivity of diatoms in 100% of cases. The negativity of diatoms in drowning deaths might be due to low abundance of diatoms in drowning medium, destruction or loss of diatoms during processing or inappropriate organ samples used for the study. ...
... In addition to the numbers of diatoms, the types of diatom are also important for the diagnosis of drowning [1,20]. Generally speaking, the same genera of diatoms in the water samples and organs are useful to identify that whether the diatoms found in the tissues are actually aspirated from the water. ...
Article
Full-text available
Diatom test has been a significant tool for the diagnosis of drowning. The reliability of the diatom test is still in strong dispute in the field of forensic science because of the false-positive results. This study was designed to quantitatively compare the numbers of the diatoms in false-positive cases and true drowning cases. Diatom samples from 64 victims were used in this study: 32 cases are confirmed drowning victims and other 32 cases died from non-drowned death. Samples were subject for the diatom test those were analyzed by the microwave digestion-vacuum filtration-automated scanning electron microscopy method (MD-VF-Auto SEM method) that we developed before. The results did show that there are false-positive diatoms detected in the liver and kidney tissues of non-drowned bodies: 6/20 in liver tissues and 7/20 in kidney tissues. However, the quantitative studies showed that there are statistical differences with the numbers of diatoms in the false-positive cases and in the true drowning cases. Diatom test of single organ is difficult for us to distinguish the sources of the diatoms detected. Therefore, comprehensive analysis of multiple organs would be more useful for the diagnosis of drowning.
... Sample collection procedures were identical of those described by Hurlimann et al. 12 All the autopsy tools were previously washed with a 2 N NaOH solution and special care was taken to prevent sample contamination. 11 Samples from the right lower and left upper lobes of lung, left side of liver and kidney (approximately 200 g each), bone marrow (10 mL) and stomach content (25 mL) were collected in a 250 mL sterile plastic container. Samples were stored at À10 C until further treatment. ...
Article
The role of the investigation of diatoms' presence in organs and body fluids of an individual found dead in a liquid medium and the relevant contribution to the forensic diagnosis of drowning remain controversial. Furthermore, the absence of an exact and well-defined method for diatoms' analysis makes its study a challenging task. Considering this medico-legal problem and the absence of forensic studies on this subject in Portugal, this work aimed to determine the drowning place of dead individuals based on the analysis of diatom species found in different tissues (lung, liver, kidney, bone marrow) and stomach content. Diatom species found in biological samples were compared with those present in the liquid medium where the corpses were found. A total of 37 cases of death by drowning in Oporto metropolitan area were studied. A seasonal database of the diatom species found in Douro river estuary was built based on water samples collected at nine selected places. Diatoms' extractions were performed by a chemical method using 37% (w/w) hydrochloridric acid for the biological samples and 96% (w/w) sulfuric acid for water samples. Diatoms were found in 63% of total cases but only in lung and gastric content samples. The absence of diatoms in other organs is probably related with a quick death, which may have stopped blood circulation almost immediately, preventing diatom contamination of the other organs. A strong relationship between the diatom species found in the biological samples and those found in water samples of the respective drowning place was observed. Due to the high anthropogenic influence on the Douro estuary no significant differences were observed between the five sampling places, making it extremely difficult to determine the exact estuary location of the drowning. The importance of the creation of a diatom database of the potential drowning places (e.g., rivers, seas, lakes) becomes clear in this study. It also shows that, in cases of drowning, the collection of a water sample from the drowning place is crucial. This is the only way to allow a rigorous comparison of the diatom species in water and biological samples.
... The 'Diatom test' is a reliable method to determine the cause of death due to drowning based on diatomological interpretations about quantitative and qualitative analysis in the human body and reference water sample was used as an indicator of antemortem drowning. [26][27][28][29] Diatoms are microscopic unicellular algae belong to class 'Bacillariophyceae' and grouped under planktonic algae. They are photosynthetic having the unique structure of siliceous double shells. ...
... • Strong nitric acid is added to the sample and heated until it becomes transparent and then centrifuged to suspend diatom at the bottom. • The suspended sediment part contains diatom, which can be seen under the microscope (Ludes et al., 1994). • The general procedure must be followed to view the diatoms under the compound microscope at high magnification of the objective lens. ...
... The other reason is that more diatoms are retained in the lung tissue than the liver and kidney tissues owing to the intense breathing movements in the drowning process. There are very few discussions regarding the correlation of diatom content in organs and mediums, Pollanen's research showed that the positive rates of diatom tests in drowning cases are influenced by different seasons because of the variety of diatom numbers in drowning mediums 2,7,17 . During drowning, diatoms in the drowning medium can penetrate into the bloodstream through the alveoli of the lung, reaching distant organs, including the liver and the kidneys 5 . ...
Article
Diatoms detected in human organs have been considered as supportive evidence for the diagnosis of drowning. During drowning, the vast majority of diatoms are retained in lung tissues, and only few are transported to distant organs via the bloodstream. In this article, we quantitatively analysed diatom contents in the lung tissues, liver tissues, kidney tissues and the drowning mediums of 165 drowning death cases. The results showed that the diatom content in lung tissues was positively correlated with that in the water samples. However, there was no linear correlation between the diatom content in the liver tissues and that in the water samples. Our study also showed no linear correlation between the diatom content in the kidney tissues and in the water samples. The diatom contents in the liver tissues and kidney tissues were positively correlated, while no linear correlations of diatom contents were found between the liver tissues and lung tissues, and the kidney tissues and lung tissues. These correlations of diatom content in the organs and drowning mediums provide valuable information to interpret and analyse the quantitative results of diatom tests.
... Analysis of diatoms in the lungs, liver, spleen, blood, and bone marrow has for many years been undertaken as a confirmatory test in possible downing cases. Pollanen [5] however contradicted the statement for lungs, which many times gave false positive test in bodies exposed to water or drowning medium Abbreviations: COI-5P, cytochrome C oxidase subunit 1-5 phosphate; psaA, photosystem I P700 chlorophyll a apoprotein AI; rbcL, ribulose carboxylase/oxygenase large subunit; rbcl-3P, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit for long time supported by many other researchers [4,6,7]. He proposed that any ambiguity regarding the diatom evaluation can be ruled out on the bases of their genomic studies, especially DNA, because it occupies a severe and very significant place in various fields of the study and bimolecular evaluations that might prove to be beneficial for the future growth of the diatom test for drowning. ...
Article
Diatoms are unicellular microalgae with cell wall made up of rigid silica found in all open water bodies. They thus resist degradation and hence are important tool to diagnose cause of death in drowned bodies. The nitric acid digestion method practiced conventionally in forensic science laboratories has limitations due to manual error. Plant chloroplast genes found in diatoms such as ribulose‐1,5‐bisphosphate carboxylase oxygenase (rbcL‐3P) and rbcL, universal plastidic amplicon (UPA), and photosynthesis I P700 apoprotein chlorophyll Al (psaA), which play an important role in photosystems I and II of photosynthesis, are tested to diagnose drowning in experimental mice. It was seen that psaA‐2 showed amplification at 150 bp in all biological samples. The sequences of psaA‐2 gene marker showed 100% proximity to Thalassiosira weissflogii and rbcL‐3P showed 99% resemblance to Pseudo‐nitzschia multiseries. On the other hand, in postmortem drowned biological samples, the chloroplast‐based gene marker failed to show any amplification.
... Ludes et al. treated organs samples with chemical digestion by using nitric acid, enzymatic digestion by using proteinase K and ashing method. Ludes et al. reported that enzymatic digestion method was more rapid, safe and environment friendly but this method takes eight hours for digestion of liver, lung, kidney and brain samples [23]. The modified reverse aqua regia method was very economical, less time consuming and environment friendly as compared to other methods such as H 2 SO 4 , nitric acid, chemical and enzymatic digestion using nitric acid and proteinase K, enzymatic digestion with proteinase K, papain, HCl, rapid enzymatic digestion, nitric acid plus hydrogen peroxide, nitric acid in disorganization can and toluene-350 method. ...
Research
Background: The presence of diatoms in human tissues contributes significantly in determining the mode of death in drowning. Conventional acid digestion of tissues for extraction of diatoms is a time consuming and potentially hazardous technique. The various techniques of extraction and analysis affect the identification of diatoms from human tissues. We have developed and tested a new method which is a simple, safe, time saving, economical extraction technique and extracts diatoms rapidly by removing the extracellular and intracellular organic matter from the siliceous frustules of diatoms. The method was compared with other conventional methods of extraction. This method provides results within 2-3 hours from the tissue samples of suspected drowning cases without interfering with microscopic observation and may be useful in current forensic practices. Materials and methods: The lung tissue samples and bone marrow from bones (femur and sternum) of 66 human cases suspected for drowning death were taken. The human tissue samples of humans were analyzed by the modified reverse aqua regia digestion method and the conventional acid digestion method simultaneously. Results: The results showed that the modified reverse aqua regia digestion method was less time consuming having very strong digestive ability with less impurity as the structure of diatoms remained almost intact, diatoms were identified with clear striations and the recovery rate was higher as compared to conventional acid digestion method. Conclusion: The modified reverse aqua regia acid digestion method is a new scientific approach in the field of forensic diatomology as this is simple and rapid procedure that produces more effective
... In recent years, considerable research activities have been focused on and significant progress has been achieved in developing better methods for preparation of diatom frustules. Several methods have been developed, which include the microwave digestion vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM) [46], chemical digestion with nitric acid plus hydrogen peroxide [47,48], enzymatic digestion [49,50], solubilization with Soluene-350 [51]. In the MD-VF-Auto SEM method, tissues are dissolved in strong acid (nitric acid + H 2 O 2 ) with the assistance of microwave. ...
Article
Diatom test is the most commonly used method to diagnose drowning in forensic laboratories. However, microscopic examination and identification of diatom frustules is time-consuming and requires taxonomic expertise. At present, the identification of drowning is still a challenge in forensic casework. In this study, we developed a novel diatom microarray based on the detection of specific 18S rRNA gene fragments of diatom species. The array covers 169 diatom species which were documented as commonly found in a wide range of fresh waters in China. Diatom arrays were prepared from species specific oligonucleotide probes targeting to variable regions of the 18S rRNA gene. We also developed an auxiliary sample preparation method for isolation of diatom DNA from tissues, which enabled detection of diatom species in real forensic samples as well as environmental waters. We applied the diatom arrays to analyze six drowned cases and eight environmental samples. The diatom arrays showed much better sensitivity and more consistent results than those of the conventional SEM methods. We discovered major discrepancies between results generated by the diatom arrays and the routinely used SEM based diatom tests. We verified the results of our diatom arrays by species specific PCR and Sanger sequencing and found that the currently used SEM diatom test method has a serious deficiency in sensitivity due to high loss rate of frustules in the sample preparation procedure. We anticipate that the application of diatom arrays will transform current forensic practice of diagnosing drowning deaths.
... Ludes et al. treated organs samples with chemical digestion by using nitric acid, enzymatic digestion by using proteinase K and ashing method. Ludes et al. reported that enzymatic digestion method was more rapid, safe and environment friendly but this method takes eight hours for digestion of liver, lung, kidney and brain samples [23]. The modified reverse aqua regia method was very economical, less time consuming and environment friendly as compared to other methods such as H 2 SO 4 , nitric acid, chemical and enzymatic digestion using nitric acid and proteinase K, enzymatic digestion with proteinase K, papain, HCl, rapid enzymatic digestion, nitric acid plus hydrogen peroxide, nitric acid in disorganization can and toluene-350 method. ...
Article
Full-text available
Background: The presence of diatoms in human tissues contributes significantly in determining the mode of death in drowning. Conventional acid digestion of tissues for extraction of diatoms is a time consuming and potentially hazardous technique. The various techniques of extraction and analysis affect the identification of diatoms from human tissues. We have developed and tested a new method which is a simple, safe, time saving, economical extraction technique and extracts diatoms rapidly by removing the extracellular and intracellular organic matter from the siliceous frustules of diatoms. The method was compared with other conventional methods of extraction. This method provides results within 2-3 hours from the tissue samples of suspected drowning cases without interfering with microscopic observation and may be useful in current forensic practices. Materials and methods: The lung tissue samples and bone marrow from bones (femur and sternum) of 66 human cases suspected for drowning death were taken. The human tissue samples of humans were analyzed by the modified reverse aqua regia digestion method and the conventional acid digestion method simultaneously. Results: The results showed that the modified reverse aqua regia digestion method was less time consuming having very strong digestive ability with less impurity as the structure of diatoms remained almost intact, diatoms were identified with clear striations and the recovery rate was higher as compared to conventional acid digestion method. Conclusion: The modified reverse aqua regia acid digestion method is a new scientific approach in the field of forensic diatomology as this is simple and rapid procedure that produces more effective results. Keywords: Drowning; Diatom Test; Digestion Method; Diatom Extraction; Modified Reverse Aqua Regia; N: Number; WHO: World Health Organization; NCRB: National Crime Record Bureau; DNA: Deoxyribonucleic Acid; PCR: Polymerase Chain Reaction; SEM: Scanning Electron Microscope.
... Acid digestion methods using nitric or sulfuric acids are widely applied in forensic casework. Other modified chemical digestion methods use enzyme [4,5], or Soluene-350 [6,7]. After chemical digestion and centrifugation, diatoms on tissue smears are then manually observed under light microscopy. ...
Article
Diatom examinations have been widely used to perform drowning diagnosis in forensic practice. However, current methods for recognizing diatoms, which use light or electron microscopy, are time-consuming and laborious and often result in false positive or negative decisions. In this study, we demonstrated an artificial intelligence (AI)-based system to automatically identify diatoms in conjunction with a classical chemical digestion approach. By employing transfer learning and data augmentation methods, we trained convolutional neural network (CNN) models on thousands or tens of thousands of tiles from digital whole-slide images of diatom smears. The results showed that the trained model identified the regions containing diatoms in the tiles. In an independent test, where the slide samples were collected in forensic casework, the best CNN model demonstrated a performance competitive with those of 5 forensic pathologists with experience in diatom quantification. This pilot study paves the way for future intelligent diatom examinations; many efficient diatom extraction methods could be incorporated into our automated system.
Article
Low rates of diatom positivity in the closed organs of drowning victims present challenges for diatom testing. High positivity rates in closed organs of non-drowning victims also raise an important issue. These contradictory findings were common in diatom testing studies undertaken during the 1960–80 s, but the reasons remained unclear. In the present study, we identified one of the most important factors associated with false-positive results in diatom testing using strong acid. One to 290 false-positive diatoms were found in reused Kjeldahl flasks that were thoroughly washed after the first diatom testing and kept free of tissue before the second testing. False-positive results occurred in 11 of 20 cases when more than approximately 10,000 diatoms were present in digested tissue or water samples. Reused flasks were found to contain many common diatoms (<30 µm), including Cocconeis, Cymbella, Diatoma, Gomphonema, Navicula, and Nitzschia, in agreement with reports of diatoms identified in closed organs. Surprisingly, such false-positive results occurred even at the sixth diatom testing using the same flasks kept free of tissues in each analysis. In contrast, no diatoms were detected in any reagent or associated with other glassware. Thus, reuse of Kjeldahl flasks can readily cause false-positive results that cannot be completely prevented by cleaning the flasks using alkali detergents, as evidenced by detection of diatoms even after six tests. We assume that diatoms causing false-positive results are partially melted by heating and fixed onto the flask’s inner surface glass, as the diatom frustule consists primarily of SiO2, similar to glass. Adherent diatoms are then released from the glass by re-heating at the next diatom testing. These results also suggest that the number of diatoms remaining in a flask can increase steadily as a result of repeated reuse for analysis of lung or water samples. In contrast, in analyses using only new flasks, only one or two diatoms were found in 4 of 20 kidney, 2 of 12 liver, and 2 of 8 blood samples from 20 drowning victims. It is difficult to determine whether such diatoms are actually carried via the blood circulation, as contamination with a few diatoms can occur during autopsy procedures and diatom testing. In conclusion, only new (unused) Kjeldahl flasks should be used for diatom testing with strong acid digestion. Moreover, these data suggest that the number and frequency of diatoms present in closed organs of drowning victims may be much lower than previously thought.
Article
Objective To determine the efficacy of using sodium hypochlorite (NaClO, Purelox) as a simple and rapid alternative digestion method for the diatom test through a quantitative comparison with the conventional method that uses fuming nitric acid (HNO3). Materials and methods In experiment 1, using 30 water samples, the NaClO and HNO3 methods were compared using paired t-test. In experiments 2 and 3, we included blank human lung plus 13 water samples and total of 32 drowned human lung samples, respectively, to compare the NaClO and HNO3 methods using paired t-test. The relationship between the concentration ratio and background factors was tested in experiment 3. Welch’s t-test was used to determine differences in the ratio between the lung side and sex, whereas Pearson’s correlation coefficient was used to determine the correlation between the ratio and either age or postmortem interval. The geometric mean of two counts was used for each specimen and all counts were logarithmically transformed to base 2 in the statistical analysis. Results The NaClO method was completed within 80 min for any sample. In experiment 1, there was no significant difference between the NaClO and HNO3 methods using water samples (the mean of the ratios: 0.99, 95% confidence interval (95%CI: 0.89 - 1.10, P = 0.80). In experiment 2, the count of the NaClO method was lower than that of the HNO3 method using lung plus water samples (the mean of the ratios: 0.47, 95% CI: 0.35 - 0.65, P = 0.0002). In experiment 3, the concentration of the NaClO method was lower than that of the HNO3 method using drowned lung samples (the mean of the ratios: 0.28, 95% CI: 0.20 - 0.38, P < 0.0001). A weak correlation between the postmortem interval and the ratio of the two methods was observed (r = -0.58, P = 0.012), although no difference between lung sides or sexes were detected (P = 0.87 and P = 0.50, respectively) and no correlation occurred between age and the ratio (r = 0.15, P = 0.43). Conclusion Using NaClO as a simple and rapid digestion method for diatom testing of water samples would be an excellent alternative to conventional methods. Although the method’s diatom detection rate for the lung samples was not optimal, it was still shown to be a feasible method.
Article
Background In forensic medicine, the diatom test is used to diagnose drowning. Drowning and postmortem immersion can be distinguished by calculating the ratio of diatom concentration in the lungs and drowning water (L/W ratio). However, this claim was based on the unproven hypothesis that diatoms may be concentrated in the lungs due to respiratory movements. This study was conducted to examine whether the L/W ratio increased with experimental water injection. Methods A total of two experiments was performed using 22 non-drowned cat carcasses found on dry land. First, for the experimental postmortem immersion, we soaked seven whole-body cat carcasses in pond water for an hour. Second, the pond water was experimentally injected one or ten times into each harvested lung from seven and eight cats, respectively. In the diatom test, two diatom species (Aulacoseira ambigua and Discostella asterocostata) that were dominantly observed in pond water as well as other diatom species were counted separately. The L/W ratios of each cat were calculated. Univariate linear regression analysis was performed to demonstrate the association among L/W ratios and the three categories of the experiments. The L/W ratios of the two experiments were compared with those of drowning or postmortem immersion cases of humans or cats. Results It was revealed that the clear L/W ratio differences between the three groups (experimental postmortem immersion<0.02, 1-injection < 0.2, 10-injection > 0.9 for all diatom counting) were with statistically significant as proven by the univariate regression analyses. In actual cases of cats and humans, L/W ratios were>0.4 for drowning and<0.04 for postmortem immersion. Conclusion The L/W ratio increased with multiple experimental water injections into the lungs, thereby verifying the validity of the diatom concentration test to diagnose drowning. The diatom test can be used to distinguish between drowning and postmortem immersion in humans and cats by calculating the L/W ratio.
Article
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Diatoms are a group of unicellular algae that have been recorded and classified for over 200 years and have been used in a range of applications in forensic field. Diatom analysis is a valuable tool in forensic field and it is useful in diagnosis of drowning cases. The basic principal of the "diatom test" in drowning is based on correlation between diatoms present in the medium where the possible drowning took place and inhalation of water causes penetration of diatoms into the alveolar system and blood stream. These diatoms got deposited into the brain, kidneys and other organs. For solving of drowning cases, hard bones like sternum or clavicle as well as soft tissues (lungs and liver) of drowned bodies and sample of water in which possible drowning take place are usually sent to the Forensic Science Laboratories for detection of diatom. A retrospective study was conducted in the department of Forensic Medicine and Toxicology, G.G.S. Medical College, Faridkot which included all cases brought by police referred from Faridkot district and nearby districts to department from a period of October 2012 to October 2014 with alleged history of drowning. During this period a total of 100 cases were brought by police to the department of Forensic Medicine and Toxicology, G.G.S. Medical College, Faridkot. The male to female ratio of the cases was 5.25:1. The most common age group was 20-40 years for males as well as females. Thirty eight percent victims were from rural background while twenty seven percent victims were of urban background. Background of rest of the cases was unknown. Diatom test was found to be positive in 38 cases while it was negative in rest of the cases.
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Forensic botany is one of the subbranches of forensic biology, which plays a crucial role in the investigation of cases related to drowning. The frequency of drowning cases remains very high, particularly in the areas where there are large number of water bodies like ponds, lakes, rivers, and canals. They provide scope for the homicidal, suicidal, and accidental drowning. A very significant evidentiary clue, which can confirm that the death is due to drowning, is the population of diatoms. Among the other important findings in drowning cases, the presence of diatoms in the vital body organs of a drowned deceased is not only a sign of drowning but also plays an important role in deciding whether the death is due to antemortem or postmortem immersion. Diatoms are not always present in all the drowning cases but if they are present (particularly in organs like bone marrow of the sternum and femur) in abundance they can confirm antemortem immersion as a cause of drowning. Earlier, the utility of diatom test to diagnose the drowning cases was debated and challenged, but various studies have shown it to be one of the better evidences in drowning cases. The use of diatomology in forensic science contributes significantly not only in determining the mode of death but also in determining the site of drowning.
Article
Pink teeth can occur in the living or as a postmortem phenomenon. In the context of forensic pathology, pink teeth are most commonly found in asphyxial deaths like drowning and strangulation; sudden and unnatural deaths, drug intoxications and carbon monoxide toxicity. However, their pathognomonic value is still doubtful as there is no obvious connection between the cause of death and this non specific phenomenon. The surrounding humid or wet environment appears to be essential in their formation. The process behind pink staining is hemolysis of erythrocytes and subsequent diffusion of hemoglobin pigment and/or its derivatives into the dentine tubules. A time delay is suggested between death and the occurrence of pink phenomenon. Two cases of putrefied corpses are described who died due to drowning and ligature strangulation, respectively. The autopsy revealed pink coloration of all the teeth in both cases which was more marked at their neck and cementum-enamel junction. The current view on the underlying chemical process, relation to the cause of death and the factors affecting the pink teeth phenomenon are explained with the help of both cases.
Chapter
This chapter contains sections titled: Introduction Postmortem Forensic Counter Measures Differences in Drowned Victims vs Those that Die of Other Causes Techniques to Identify Diatoms in Biological Sample Case Studies Identification of Diatom Using Molecular Tools in Tissue and Water Samples Differentiation of Diatom DNA in the Tissue of a Drowned Victim Polymerase Chain Reaction (PCR) Diatom DNA Extraction from Biological Samples of a Drowned Victim Best Barcode Markers for Diatoms to Diagnose Drowning DNA Sequencing Advancement in Sequencing Leads to Advancement of Data Interpretation Conclusion and Future Perspectives Acknowledgements List of Abbreviations Used
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Background: Diatoms found inside the body of a drowned victim may serve as corroborative evidence in the diagnosis of cause of death. Diatom has proved to be the only golden standard for diagnosis and confirmation of drowning deaths whether the drowning was ante-mortem or post-mortem. Methods: The study was based on the cases of death due to drowning received from three districts of Northern Range of Himachal Pradesh, India during the period of five years from I st January, 2010 to 31 st December, 2015 for diatom test. A total of 66 human cases were examined for detection of diatoms. The detailed information regarding cause of death, socio-demographic factors and other associated information was gathered. The acid digestion method accepted worldwide for diatom extraction was used. Results: Male victims predominated (75.75%). Most common affected age group was 21-40 years (53.02%). The youngest victim was a girl of 4 years age who drowned accidentally in a water tank, while the oldest victim being a 86 years old who fell accidently in a river. Married victims contributed to 33 (50.0%) and unmarried to 21 (31.81%). 13 (19.69%) drowning cases belonged to students followed by labourers (18.18%) and housewives (15.15%). The highest reported cases of drowning were from the rural areas (65.15%) followed by urban areas (16.66%). Majority (81.81%) of the victims drowned in fresh water. The incidences of drowning were more in water of natural flowing streams (khuds) (31.81%) followed by rivers (22.72%), nullahs/rivulets (12.12%), wells (9.09%), kuhls/water channels (7.57%), canals (6.06%), ponds (3.03%), water tanks (3.03%) and check dam, dam and waterfall in one each (4.54%) respectively. Accidental drowning was the most common cause of death (37.87%). Highest (51.51%) percentage of drowning cases was noticed during the months of monsoon/rainy season. Blood on mouth and nostrils was present in 14 (21.21%), froth from mouth, nose, larynx and trachea in 35 (53.03%) cases. Diatom-test was found positive in 62 (93.93%) cases. The results of the study revealed the occurrence of various varieties of diatoms in water bodies of northern region of Himachal Pradesh. The most common diatom genera detected were Navicula (86.36%). Conclusion: The study concluded that diatoms are amongst the important biological forensic evidences in diagnosing the cause and place of death due to drowning. Gender based examination revealed higher percentage of males involved in drowning fatalities and the accidental submersion was the commonest manner of death. Diagnosing Death with Diatoms: A Retrospective Study of Forensic …
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Diatoms are microscopic, eukaryotic and unicellular algae that are abundant in most aquatic habitats and are useful proxies for the ecological analysis. They are used as a tool to differentiate the ante-mortem drowning cases from post-mortem submersion. In the present study, water samples were collected from different water bodies of Indore region, Madhya Pradesh to generate the data regarding different drowning associated diatoms species found in them. Among the different diatoms genus, Navicula and Nitzschia were most common, followed by Synedra and Gamphonema. The Diatomological Mapping (D-Mapping) of water bodies might be of vital forensic importance as it can give useful lead to the forensic pathologists in solving the drowning cases, particularly when reference water sample is not available.
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Road traffic accident is one among the top five causes of morbidity and mortality in South-East Asian countries including India. Its socio-economic repercussions are a matter of great concern. This present study has been conducted to assess the prevalence and epidemiological factors related to road traffic accidents and its fatalities. Victims with alleged history of road traffic accidents, who were admitted to Dr. Prabhakar Kore Hospital and Research Centre, Belagavi and subsequently died and underwent autopsy were included in the present study. A total of 1300 medico-legal autopsies were conducted during the period, of which 642 were road traffic fatalities, constituting nearly 50% of the total cases. Road traffic accidents are a major cause of death among all un-natural deaths. Our study intends to find out the epidemiological factors, risk factors, use of safety measures, compliance with traffic rules and regulations, presenting injuries and interventions required in victims of road traffic accidents to prevent morbidity.
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Algae are eukaryotic photosynthetic organisms found in freshwater, marine, or brackish water. They are capable of surviving in harsh conditions such as in snow, deserts, hot water spring, and in mutual relationships with other organisms like lichens, making them as cosmopolitan species in environment. Algae play vital role in every aspect may it be as primary producers in food chain or fixing maximum amount of carbon dioxide in nature. But talking about applications of algae in this paper, we have focussed on Diatoms (class Bacillariophyceae) which are golden brown algae. Their ubiquitous presence, small size, and silicious frustule with peculiar ornamentation add uniqueness to their identity. Nowadays diatoms are considered as prime evidence in solving cases of drownings in the fields of forensics. Diatoms from the drowning site can tell about the physicochemical nature of environment and help in knowing the mode of death of a drowned victim. As during drowning, these small entities enter inside the body through body openings and once penetrated inside the body they get circulated and deposited in various organs till the victim dies. These penetrated diatoms are then recovered as evidence by various techniques and then their microscopic studies further help in their identifications. This helps in linking the crime scene and the victim during the investigation and thus helping the crime mystery of drowning to be solved. Because of their vast role in the drowning case investigation they are regarded as golden standards in the fields of forensics.
Article
Diatom analysis is very effective for positive diagnosis of water inhalation in drowning. However, conventional strong acid diatom testing is laborious and potentially dangerous. We propose a simple, fast, and safe protocol using inexpensive reagents such as papain, SDS, and 5 N HCl for extracting diatoms from lung, kidney, and liver tissues. First, we determined optimal conditions for papain digestion using porcine tissues. Papain digestion was clearly superior to Proteinase K digestion. Next, for assessing the assay effectiveness in practical cases, the papain digestion protocol was applied to 80 tissue samples from 20 suspected drowning victims. Left and right lung tissues (1 g each) were digested in 15-mL conical centrifuge tubes. Kidney and liver tissues (10 g each) were extracted in 175-mL conical centrifuge bottles. Papain dissolved all organs sufficiently and permitted clear visualization of diatoms, although papain's solubilization activity was still inferior to strong acid digestion. The proposed enzymatic method requires only a low-speed centrifuge and water bath. Diatoms typically can be extracted from tissue samples within 3–5 h. The cost of protease is reduced some 6-fold by using papain in place of Proteinase K. Thus, the proposed method can be useful as a less-laborious, less-hazardous, and less-costly minimal test when the conventional strong acid digestion method is not performed due to personnel, equipment, budgetary limitation, or environmental and safety considerations.
Article
Diatoms also called as the ‘jewels of sea’ are microorganisms which are extensively found in the aquatic system. These unicellular organisms make up nearly half of the biological material in the water body. It is also one of the most significant biological evidence that is obtained in case of drowning. The diatoms that infiltrate inside the body of the deceased may serve as a corroborative or even conclusive evidence to support the diagnosis of death. These diatoms also help in ascertaining whether the drowning is ante-mortem or post-mortem. The review discusses the current extraction procedures and microscopic examination techniques used in forensic science for diagnosis of death by drowning.
Chapter
Recovery of a body from water does not mean drowning. Other causes of death, including trauma, are possible. Drowning is a type of asphyxia that involves complex pathophysiological mechanisms, which contribute to death. Premorbid conditions (e.g., ischemic heart disease, epilepsy, ethanol intoxication) increase the risk of drowning. Despite the complexity of the drowning process, associated external and internal findings are few. The lack of specific signs and submersion artifacts can hinder the determination of the cause of death in a presumed drowning. Various drowning “tests” have been used to assist in this assessment. A bathtub death can be particularly challenging because all manners of death occur. A diver’s death, ironically, can be caused by the artificial air supply.
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The authors have once again examined the so-called diatom test from the point of view of its evidential value, and they have come to the conclusion that the number of diatoms in the femur of non-drowning victims is as high as that in the femur of drowning victims. In this connection they cite in the first place Otto (1961) who is said to have also detected diatoms in the great-circulation organs of non-drowning victims. This is not true. Otto examined normal lung dust and found in a strikingly systematic way diatoms, diatom fragments, and other organic siliceous skeletons. It was Krauland who arrived at the idea of testing for diatoms on a wide scale, and these investigations were carried out within the framework of a dissertation. Although this was not published until 1965 (Schneider), partial results were presented earlier by Spitz at meetings in Mnster (1962) and London (1963) and published by Spitz and Schneider in 1964. Even if there is continuing discussion about the value of the diatom test as evidence, it can at least be stated at this time that the test is definitely not appropriate for the differential diagnosis of drowning death/sudden death from natural causes in the water.
Article
The determination of the cause of death of a person whose body is found in the water requires careful investigative work on the part of both police and pathologists. Opinions as to cause and manner of death should not be derived from the autopsy alone but must result from a logical correlation of data regarding victim identity, circumstances, autopsy laboratory findings.
Article
An improved method for detection of diatoms in case of drowning is reported. The sample is digested with nitric acid. In order to avoid destructions of diatoms as well as losses by centrifugation etc. the time for wet digestion was reduced and the digest solution is filtered through a membrane filter. Fatty material is removed by alternate washing the filter with 2-Propanol and Petroleum ether. Following the wet digestion of the filter, aliquots of this digest solution are filtered and the filter is examined microscopically. As the recommended procedure is less time consuming and yields almost complete recovery of diatoms the method has great probative value in case of death by drowning.
Article
Diatoms in the bone marrow (femur) of 16 nondrowned bodies were detected by a modern method of ultrafiltration. The rate of diatoms were found to be in the same range as in cases of drowning. The results deny the proof of diatoms even in bone marrow to be useful any longer for the diagnosis of death from drowning.
Article
With the aid of experiments in animals we have again investigated the question as to the quantitative and qualitative immigration of diatoms into the greater circulation and thus into the bone marrow at the time of drowning. We have applied an experimental method which meets this complex topic. Thus, just before the animals (rabbits) were drowned, one of their hind legs was amputated, and the vessels of one of their kidneys were clamped to learn how many diatoms were present before the drowning. After the drowning, a qualitative and quantitative examination of the bone marrow of both hind legs and of both kidneys was carried out. In all cases, an increase in the number of diatoms could be recorded as compared to the initial number of diatoms present prior to the drowning. Based on the results of the experiments and the information gained in the examinations criteria are discussed which, when applied, allow positive diatom findings to indicate valuable diagnostic information about death by drowning. Reference is made to preservation of the death causing liquid in the stomach.
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The occurrence, methods of treatment, and applications of diatoms in forensic science are described. Reference is made to drowning incidents, and other cases where the use of diatoms by the forensic scientist can provide valuable evidence.
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Death by 'drowning' is classically considered asphyxial in nature although there is frequently little evidence to support this concept at postmortem examination. The results of the use of two simple tests: estimation of plasma specific gravity of heart blood and magnesium concentration of vitreous humour, as an aid to the scientific assessment of the cause of death of deceased persons found dead in salt and fresh water are given. The role of prior consumption of alcohol in deaths by immersion is discussed.
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According to the studied material the majority of the bodies found submersed in water (in the area covered by this report) were predominantly those of Caucasian males between 20 and 40 years of age, probably sober and without drug consumption when they entered the water. The majority of the cases were recovered from the water in the late spring and early summer and many of them were in different stages of decomposition which made the diagnosis of drowning impossible. The diagnosis of the cause and manner of death in such cases requires a complex interpretation of all investigative evidence and a logical approach in each case in particular.
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Since the 1950s, quantitative diatom analysis has been used successfully at the Department of Forensic Medicine at the University of Helsinki as a supportive method for diagnosing deaths by drowning. The reliability of the method was firmly established in 1986 by a study involving 107 probable cases of drowning. Since 1982, the quantitative analysis has been complemented with qualitative diatom analysis. This report presents potential applications of the latter method by describing its use in six cases of drowning.
Article
Bodies found in water may cause problems for forensic pathologists who have to differentiate drowning from postmortem immersion or fresh from salt water drowning. The exact physiopathology of drowning is still controversial and complementary tests can not exactly establish the exact cause of death if macroscopic findings at autopsy are not conclusive. We have employed atrial natriuretic peptide (ANP) as a marker in an experimental series of fresh and salt water drowning, comparing their results with a non-drowned control series. There are differences between the plasma basal levels of the control series (79 pg/ml) and the levels in animals drowned in fresh water (358 pg/ml, P less than 0.001) and between control and rabbits drowned in salt water (190 pg/ml, P less than 0.001). According to these values, there are also differences between fresh and salt water drowned animals (P less than 0.001). We propose this peptide as a new marker in cases of drowning, with the ability to differentiate drowning from postmortem immersion and fresh from salt water drowning.
Article
This study investigates drownings in Uusimaa province in Finland in order to form a picture of the people who end their lives in this way. The material consisted of 285 successive drownings, which were subject to autopsy at the Helsinki University Department of Forensic Medicine between 1978 and 1986 and for whom diatom analysis was carried out. There were 51 definitive cases of suicide by drowning. For these, an analysis was made of age and sex distribution, alcohol and drugs, scene of the incident, significant diseases, social group, place of residence, information on suicide notes, previous suicide attempts, treatment for mental problems, depression preceding the suicide, talk of suicide and heavy use of alcohol. According to this material the typical Finnish drowning suicide is a man from 21 to 30 years old, who belongs to the lowest social group, is unmarried, and lives in Helsinki. He drowns sober during June in the shallow water off the coast of the Gulf of Finland, has previously attempted suicide, received treatment for mental problems, and has been depressed recently.
Article
In order to corroborate the medico-legal diagnosis of vital submersion we reviewed the appropriate literature and tested Icard's initial hypothesis, already expressed in 1932, that Strontium might be a good indicator of sea-water drowning. Therefore, we examined all the bodies found either in fresh or in sea-water, and also non-drowned control cases. Strontium concentrations in both cardiac cavities and in a peripheral blood-vessel were determined by flameless atomic absorption spectrophotometry. The statistical evaluation of the results is hereby presented. We come to the conclusion that this determination can be a valuable additional proof for sea-water drowning diagnosis, whilst freshwater drowning would be more difficult to assess. Further investigations are being undertaken.
Article
An examination is made of the applicability of quantitative and qualitative diatom analysis to the diagnosis of death by drowning, definition of the environment in which drowning occurred, and delimitation of the area where it occurred. The material comprises 107 bodies of subjects known or suspected to have died by drowning together with a control series of 15 bodies of subjects over 30 years of age who had died of various diseases on land. Whenever diatoms were found in the greater circulatory organs they were also found in the lungs, and when none were present in the lungs none were found in the other organs either. No diatoms or fragments of diatoms were found in the samples from the control subjects. All the fresh, well-preserved bodies for which death by drowning could be regarded as certain from the macroscopic autopsy findings and police reports, the cases used to test the method, gave quantitative diatom results that supported a diagnosis of water aspiration. The diatoms identified in the qualitative analyses served well to describe the ecological properties of the environments in which death had taken place, and the site of drowning could be defined by means of comparative water samples provided that sufficient diatoms were present, the local environment was not too homogeneous or the diatoms were not of quite different species due to a completely unknown location of death.
Article
Levels of magnesium, potassium, sodium and calcium in post-mortem vitreous humour from human controls, fire fatalities and drowning victims have been determined. The effects of time-related internal changes, external environmental parameters and different causes of death are evaluated. Despite the positive correlation and marked increase of potassium and, to a lesser extent, of magnesium and calcium with the length of the post-mortem interval, individual biological variability severely limits the usefulness of predictions of post-mortem interval based on electrolyte metal data. At best, there is only a 2/3 chance of a prediction being within 12 h of the true value. Vitreous humour metal concentrations are affected by external influences, such as the elevated temperatures of fires which increase the rate of release of intracellular magnesium and potassium. In cases where drowning is suspected, establishment or exclusion of this cause of death is not possible on the basis of vitreous humour electrolyte metal data because of possible post-immersion diffusion across the permeable membrane of the eyeball. It appears, however, that magnesium in salt-water cases and sodium in fresh-water cases are related, albeit erratically, to the length of the immersion period.
Article
The author first considers what is known at present of the physiopathology of drowning.Subsequently full attention is given to obvious differences between experimental drowning and drowning as it normally occurs. Putrefaction and post-mortem mutilation are both frequent and set limits to the investigation possibilities of drowned bodies. The picture is altered in relation to the degree of pollution of the water responsible. Moreover resuscitation efforts, i.e. artificial ventilation and drug administration, can influence some post-mortem laboratory measurements.Hydrocution and death from hypothermia in water are then commented upon. These cases call rather for a diagnosis per exclusionem, since no visible specific cadaveric signs are induced, except for the occasional diatom penetration into the lungs.The investigation possibilities under these different circumstances are summed up.The possible findings in the upper air passages and stomach are considered in regard to their diagnostic significance. Most of the methods applicable to examination of lung tissue are mentioned. They are judged according to their own merits. Blood analysis in relation to the aqueous inflow or biochemical reactions are summarized. The diagnostic value of the presence of diatoms in the organs, and particularly in the bone marrow, is sketched and illustrated by two particular cases.In conclusion the author emphasizes the reliability of the diatom examination when used critically in experienced hands.
Article
The content of diatoms in 5 samples: lung-, kidney- and liver-tissue plus columna- and femur-marrow from each of four drowned and four non-drowned persons has been investigated. Diatom valves were found in all the samples. It seems, however, impossible to point out any characteristic differences between the composition of the diatom 'flora' in drowned and non-drowned persons. Consequently it will not be possible by means of diatoms to prove that a person died by drowning.
Article
The use of diatoms in the diagnosis of cause of death by drowning is reviewed. The arguments for and against the method are described, and the possible reasons for divergent opinions explored. Attention is drawn to the observations of some workers that the organs of non-drowned subjects may contain diatoms. Important papers in Hungarian, German, Russian and French particularly relevant to this subject have been translated and their essence presented here.
Article
A new and simple method for diatom detection is presented. Organ tissue was digested by a solubilizer instead of a conventional acid which may be hazardous and pollutive. The digestion was accelerated by ultrasonic irradiation and heating.
Article
Studies are reported on the enzymatic digestion method for detection of plankton from lung tissue by using proteinase K with sodium dodecyl sulphate. This method is simple, safe and effective for detection of not only phytoplankton including diatoms but zooplankton which are destroyed by the acid digestion method. The present method is, therefore, much more advantageous for diagnosis of drowning than the disorganization method using strong acids.
Diagnostik des Ertrinkungstodes und Bestimmung der Wasserzeit
  • Reh
Detection of diatoms in the bone marrow of non-drowning victims
  • Schneider
Schneider V (1980) Detection of diatoms in the bone marrow of non-drowning victims. Z Rechtsmed 85 (4) : 315-317
Drowning. In: The essentials of forensic medicine
  • Cj Poison
  • Dj Gee
  • Knight
Poison CJ, Gee DJ, Knight B (eds) (1985) Drowning. In: The essentials of forensic medicine. Pergamon Press, Oxford, pp 421-428
Zur Diatomeen Assoziations Methode: Alt-bekanntes "neu" entdeckt?
  • Schneider
Schneider V (1990) Zur Diatomeen Assoziations Methode: Alt-bekanntes "neu" entdeckt?. Z Kriminalistik 44:461
Diatoms and drowning Suicide by drowning in Uusima Province in Southern Finland Quantitative diatom analysis as a tool to diag-nose drowning
  • A Auer
  • Mottonen
Auer A, Mottonen M (1988) Diatoms and drowning. Z Rechtsmed 101 : 87-98 2~Auer A (1990) Suicide by drowning in Uusima Province in Southern Finland. Med Sci Law 30 : 175-179 3,Auer A (1991) Quantitative diatom analysis as a tool to diag-nose drowning. Am J Forensic Med Pathol 12(3):213-218
Über den Nachweis von radioaktiv markierten Diatomeen in den Organen
  • V Schneider
  • Kh Kolb