Forensic Science International 251 (2015) 179–185

Contents lists available at ScienceDirect

Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Technical Note

Simple protocol for extracting diatoms from lung tissues of suspected

drowning cases within 3 h: First practical application§
Eiji Kakizaki *, Nobuhiro Yukawa
Division of Legal Medicine, Department of Social Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan

A R T I C L E I N F O A B S T R A C T

Article history: Conventional acid digestion of tissues for analyzing diatoms obtained from suspected drowning cases is
Received 13 June 2014 time-consuming, laborious and potentially dangerous. We propose a new protocol for solubilizing lung
Received in revised form 14 December 2014 tissue using only Qiagen Proteinase K, Qiagen Buffer ATL, and 5N hydrochloric acid that can accelerate
Accepted 24 March 2015
and simplify diatom extraction from suspected drowning cases. The lower lobe of the right lung (1 g,
Available online 7 April 2015
inner region) and the upper lobe of the left lung (1 g, peripheral region) of ten immersed victims were
digested in 15-mL conical centrifuge tubes containing 9 mL of Buffer ATL and 1 mL of 20 mg/mL
Keywords:
Proteinase K solution at 56 8C for 15–60 min. The digest was washed with ultrapure water and then
Drowning
heated at 70 8C for 15 min in 5N hydrochloric acid. The acid residue was washed with ultrapure water
Diatom test
Enzymatic digestion followed by ethanol. The identification of 60–23,000 valves in 20 lung samples from the ten victims
Tissue solubilization suggested that they had aspirated water before death. The proposed digestive protocol required only a
Proteinase K low-speed centrifuge and a block incubator (or water bath) and diatoms could usually be extracted from
Hydrochloric acid lung samples within about 3 h. Informative results of lung diatom tests were very helpful to confirm
drowning as a cause of death. Therefore, the proposed protocol can be useful as a simple minimal test to
support or challenge a diagnosis of death by drowning when characteristic autopsy findings of drowning
are obvious and the probability of drowning is very high, when characteristic autopsy findings of
drowning are absent and the probability of drowning is very low, and when conventional diatom testing
is not performed due to logistic, personnel or budgetary limitations.
ß 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Drowning is one of the most difficult causes of death to prove
post mortem [1–7], and many tests have been proposed to support
a conclusion of drowning [4,7]. Among them, the diatom test is one
of the most informative and reliable laboratory tools to support the
diagnosis of death by drowning together with characteristic
macroscopic autopsy findings such as external foam, frothy fluid in
airways and overinflated lungs, although the specificity and the
sensitivity of diatom tests have attracted criticism and controversy
[1–7].
Various organs (lung, kidney, liver, brain, etc.), as well as the
blood (cardiac blood) and/or bone marrow (femur, sternum) of
immersed victims can be tested for diatoms. In diatom-rich water,
finding diatoms in the lungs can substantiate the diagnosis of ante-
§
Presented in Part at the 9th International Symposium on Advances in Legal
Medicine (ISALM), 18 June 2014, Fukuoka, Japan.
* Corresponding author. Tel.: +81 985 85 0991; fax: +81 985 85 6406.
E-mail address: eijik@med.miyazaki-u.ac.jp (E. Kakizaki).
morten water aspiration and provide information about the nature
(sea or fresh water) of the drowning liquid [8–15]. In this
environment, the numbers and types of diatoms detected in the
lungs of drowned victims are usually abundant [8–15]. Conversely,
in non-drowned victims, diatoms are generally negligible or absent
[16–19]. Some diatoms might enter the lungs along with water
during post mortem immersion, but they would not readily enter the
periphery of the lungs except for situations such as powerful water
pressure or advanced putrefaction. In contrast, few diatoms can be
detected from enclosed organs such as the kidneys and liver,
especially when the density of diatom in the drowning media is low.
Moreover, a very low number of diatoms in enclosed organs implies
the possibility of contamination during autopsy or diatom testing, or
of samples being false-positive due to an original presence
(antemortem contamination during life unrelated to water aspira-
tion) in such organs, and these cannot be completely ruled out.
We believe that the presence of diatoms in lungs should be
assessed, as far as possible, in all immersed victims. Therefore, we
propose a new protocol for dissolving lung tissue using only
commercial reagents to accelerate and simplify diatom extraction
from suspected drowning cases.

http://dx.doi.org/10.1016/j.forsciint.2015.03.025
0379-0738/ß 2015 Elsevier Ireland Ltd. All rights reserved.
180 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185

Table 1
Features of present cases.

Case No. Discovery site Salinity of Water depth at Total or part Froth in Duration in Duration from Season Water Ambient Resuscitation
water at discovery site of cadaver mouth and/or watera body recovery temp temp
discovery (immediately immersed nose at time to autopsy (8C)b (8C)b
site (%) below head) in water of cadaver [duration (h)
retrieval of refrigeration
at 4 8C]

1 Ditch <0.05 <20 cm Partb1 No <2 h 42 h [35 h] Winter 12 11 +

2 Ditch <0.05 <5 cm Partb1 No <20 h 33 h [35 h] Winter 7 9 
3 River <0.05 2m Wholeb2 Yes <27 h 71 h [63 h] Winter 10 13 
4 Lake <0.05 60 cm Wholeb2 No <52 h 69 h [61 h] Winter 10 8 
5 River <0.05 2m Whole Yes <19 h 45 h [36 h] Spring 12 11 
6 Ditch <0.05 40 cm Wholeb2 Yes <13 h 69 h [62 h] Summer 22 28 
7 Near estuarya1 <0.05 80 cm Wholeb2 No 2–3 d* 46 h [39 h] Spring 12 12 
8 Near estuarya1 <0.05 90 cm Wholeb2 No <15 d 64 h [60 h] Winter 11 14 
9 Sea (beach)a1, a2 3.03 20–30 cm Part Yes <4 h 42 h [36 h] Winter 17 15 
10 Sea (beach)a1, a2 3.17 20–30 cm Part No <42 h 44 h [35 h] Spring 19 21 
a1
Water line changes with tides.
a2
Water edge.
b1
Water does not easily passively penetrate lungs by water pressure. However, mouth was immersed in water at time of cadaver retrieval.
b2
Almost total immersion, but cadaver did not sink.
*
Estimate.
a
Elapsed time between discovery and being reported missing.
b
Temperature at discovery.

2. Materials and methods
2.1. Autopsy cases
We obtained tissue samples from 10 immersed cadavers that
were retrieved from rivers, lakes, or ditches (cadavers 1–6),
around estuaries (7, 8) and the sea (9, 10). Table 1 shows the
profiles of each case. Cases 1, 2, 9, 10 were discovered in <30 cm
of water. Cases 4, 6, 7, 8 were retrieved from <90 cm of water and
Case 3 was retrieved from a depth of 2 m, but the body did not
sink to the bottom. Only case 5 sank to the bottom in a car to a
depth of 2 m.
The probability of drowning based only on autopsy findings
was high for all victims (Table 2). The cause of death was
finally determined in principle based on autopsy findings, as
well as the findings of two types of diatom analyses (proposed
rapid enzymatic digestion and conventional fuming nitric acid
digestion), electrolyte analysis of pleural effusions [20–23],
toxicological test results, and reliable circumstantial evidence
provided by police and other investigators.
The Research Ethics Committee of University of Miyazaki
approved this study, which proceeded according to the principles
described in the Declaration of Helsinki.
2.2. Tissue sample collection
Liver, kidneys and lungs were resected during evisceration at
autopsy. After photographing, weighing and measuring the organs,
one kidney (left or right), part of the liver (lateral one-third portion
of the right lobe), the upper lobe of the left lung and the lower lobe
of the right lung were individually placed in vinyl bags until the
autopsy was completed (hila of both lungs were clamped). After
autopsy, the surface of each organ was washed with 1 L of
ultrapure water (total 4 L) (Millipore, Billerica, MA, USA).
The capsule and peritoneum were removed from the kidney
and liver, respectively and sampled for conventional diatom

Table 2
Autopsy findings of present cases.

Case no. Partial autopsy findings Probability of

drowning based
Age (y) Height Weight Putrefactiona Froth in Mud or Fluid in Pleural Lung Lungsd (g) Lung Pulmonary
only on autopsy
(cm) (kg) airwaysb sand in airwaysb effusionc adhesiond overinflationd,e edemad,f
findings
airwaysb (ml)

1 70s 145 40  – – + L 40, R 0 Lg, Rh L 568, R 730 L++, R+ L+, R++ Very high
2 80s 161 51  – Mud, ++  L 0, R 6 Lh L 486, R 526 L+, R++ + Very high
3 80s 159 39 + – –  L 30, R 180 Lg L 594, R 634 ++ ++ Very high
4 60s 167 52   –  L 40, R 180 – L 708, R 774 ++ ++ Very high
5 50s 173 60   –  L 0, R 4 – L 816, R 922 ++ ++ Very high
6 60s 161 65   Mud,   L 110, R 60 Rg L 542, R 660 ++ ++ Very high
7 50–60s 149 38   –  L 6, R 0 Lg, Rg L 380, R 498 ++ ++ Very high
8 60s 163 48 ++ – –  L 310, R 480 – L 376, R 446 + + High
9 70s 149 46  ++ Sand,  ++ L 40, R 60 – L 460, R 504 ++ ++ Very high
10 30s 165 59 ++ – Sand, ++  L 640, R 570 – L 410, R 520 + + High
a
Degree of putrefaction is expressed as:  (none); + (slight; discoloration at abdomen), ++ (marbling).
b
Amount of froth, mud, sand, or liquid in airways is expressed as: –, none or essentially none; , small; +, moderate; ++, large.
c
L, left thoracic cavity; R, right thoracic cavity.
d
L, left lung; R, right lung.
e
Degree of lung overinflation is expressed as: –, none; +, large; ++, remarkable.
f
Degree of pulmonary edema is expressed as: –, none or essentially none; +, low or moderate; ++, high.
g
Partial.
h
Total.
 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185 181

testing. Lung samples for rapid enzymatic digestion and conven-

tional acid digestion were gathered from a wide area of each lung
lobe (Supplementary Fig. S1). All tissue samples were stored at
80 8C and analyzed within 6 months.

2.3. Proposed rapid enzymatic digestion for diatom detection

We analyzed 20 lung tissue samples obtained from the lower

lobe of the right lung (1 g, inner region) and from the upper lobe of
the left lung (1 g, peripheral region) of ten immersed victims. The
negative control (ultrapure water 1 mL) (Millipore) was simulta-
neously processed to confirm the presence or absence of
contamination arising from various reagents (Proteinase K, Buffer
ATL, HCl, ultrapure water, ethanol, etc.) whenever a new lot of
reagents was used. Lung tissues were minced using a scalpel in
Petri dishes (Supplementary Fig. S2) and then placed into 15-mL
conical polymethylpentene (TPX) centrifuge tubes (Sumitomo
Bakelite, Tokyo, Japan) with 9 mL of tissue lysis Buffer ATL (Qiagen,
Hilden, Germany) (blood remaining in Petri dishes was recovered
using 1–2 mL of buffer ATL) and 1 mL of 20 mg/mL QIAGEN1
Proteinase K (Qiagen), and digested at 56 8C for 15–60 min using a
BI-517H block incubator (Astec, Fukuoka, Japan) (Supplementary
Fig. S3). The digest was centrifuged at 2270  g for 10 min and the
supernatants were gently aspirated (Supplementary Fig. S4). The
remaining residue (about 1.5 mL) was mixed with ultrapure water
(about 12.5 mL) and separated by centrifugation. The supernatant
was gently aspirated, and the remaining residue (about 800 mL)
was mixed with about 13 mL of 5 mol/L (5N) of hydrochloric acid
(16.9%; Nacalai Tesque, Kyoto, Japan) and heated at 70 8C for
15 min. After centrifugation, the residue (about 800 mL) was mixed
with about 13 mL of ultrapure water and separated by centrifuga-
tion. Ultrapure water was added to the residue (800–900 mL) and
the weight was adjusted to 1 g using an electronic balance (A&D
Company, Tokyo, Japan) (Supplementary Fig. S5). When quantita-
tive diatom analysis is not needed, this process should be omitted.
Depending on the amount of residue (or predicted numbers of
diatoms), part or the entire residue (usually 0.1, 0.2, 0.5 or 1.0 g)
was mixed with about 13–14 mL of 99.5% pure ethanol (Guaran-
teed reagent; Nacalai Tesque) and separated by centrifugation. The
supernatants were gently aspirated, and the residues (about
200 mL) were dried on glass slides (0.1–1.0 g of lung/slide) and
mounted with Mount Media (Wako Pure Chemical Industries,
Osaka, Japan) with a refraction index 1.5 (Supplementary Fig. S6).
Fig. 1 summarizes the extraction process.
We counted diatoms in lungs using an Eclipse Ni-U with an
objective lens, a CFI Plan Apo or Plan Fluor optical microscope
(Nikon, Tokyo, Japan) at a magnification of 600 or 1000 for small
diatoms (<10 mm). Destroyed diatom valves were counted as one
diatom when over half of the original shape was retained. We
excluded diatoms from the count when only the girdle (such as the
epicingulum and hypocingulum between the epivalve and hypo-
valve) remained. Connected diatom valves were counted individu-
ally. For example, a single colony of Fragilaria in which three valves
were linked by a spine was counted as three diatom valves.

2.4. Conventional diatom testing using strong acid

We analyzed tissue samples obtained from the lower lobe of the
right lung (5 g, inner region without pleura), the upper lobe of the
left lung (5 g, peripheral region including pleura), the left or right
kidney (10 g), and the right lobe of the liver (10 g) by conventional
diatom testing. Water samples (100–1000 mL based on the
quantity of sediment) collected from locations near discovery
sites were also examined. A negative control (ultrapure water
10 mL) (Millipore) was simultaneously analyzed to confirm the
presence or absence of contamination arising from reagents
Fig. 1. Protocol for rapid enzymatic digestion.
(fuming nitric acid, hydrogen peroxide, ultrapure water, ethanol,
etc.) or the air.
The samples were transferred to 300-mL Kjeldahl flasks and
then 120 mL of 90–94% pure fuming nitric acid (Guaranteed
reagent; Nacalai Tesque) was added. The flasks were left for 12 h at
room temperature in a Draft chamber fume hood (Dalton, Tokyo,
Japan) and boiled over a gas burner for 60–90 min. The fuming
182 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185
Fig. 2. Lung tissue solubilization by rapid enzymatic digestion (right lung of Case 8).
nitric acid was repeatedly replenished until all tissue was
completely dissolved. Two or three 5-mL volumes of hydrogen
peroxide (30%; Guaranteed reagent; Santoku Chemical Industries,
Tokyo, Japan) were also added.
The solute was washed three times with ultrapure water
(Millipore) using centrifugation and finally twice with ethanol
(Guaranteed reagent; Nacalai Tesque). Residues were mounted
on glass slides and diatoms were counted as described above
(Section 2.3).
3. Results and discussion
3.1. Dissolubility of lung tissue using proposed rapid enzymatic
protocol
Qiagen Proteinase K and Qiagen Buffer ATL were originally
designed to extract DNA from tissue. Characteristically, the
working concentration of the proteinase K is 20-fold (2 mg/mL)
that in the standard method (0.1 mg/mL) of extracting DNA from
tissue [24,25], and lung tissues usually rapidly dissolve within 15–
60 min. Enzymatic digestion using proteinase K (or combined
enzymatic and chemical digestion) reported by forensic pathol-
ogists or other types of scientists also include working proteinase K
concentrations of 0.1 mg/mL, and thus tissues need to be digested
for > 20 h.
Digestion of tissue samples using Qiagen Proteinase K and
Buffer ATL proceeded as described in the manual provided with the
QIAamp1 DNA Mini kit (Qiagen) although the manual described
the small-scale extraction of genomic DNA from 25 mg of tissues
(or 0.2 mL of whole blood). We applied part of this protocol to large
scale tissue extraction (40-fold) using only Qiagen Proteinase K and
Buffer ATL. These reagents usually dissolved 1 g of lung tissue
within 15–60 min (Fig. 2a and b). However, matter (such as
connective tissue) in lung samples that sometimes remained
insoluble despite heating for >60 min was solubilized by heating
at 70 8C for 15 min in 5N hydrochloric acid (without boiling
because all reactions proceeded in 15-mL plastic centrifuge tubes;
Fig. 2c). Our purpose was not to recover DNA, thus its
decomposition did not influence the recovery of silica diatoms.
Therefore, 5N (16.9%) hydrochloric acid accelerated solubilization
when almost all lung tissue was dissolved. Moreover, 5N
hydrochloric acid at 70 8C is not as dangerous as fuming nitric
acid. Dı́az-Palma et al. [26] and DiGiancamillo et al. [27] have
described the solubilization of tissues including lung samples
using 20% hydrochloric acid, and its validity has also been
demonstrated.
The quantity of the final residue obtained from 1 g of lung tissue
using the proposed protocol was appropriate; it did not interfere
with visualization by light microscopy, and diatoms were also
clearly evident (Fig. 3).
Both proteinase K and hydrochloric acid digestion can be
accelerated by agitating samples at high speed in a vortex mixer
(Scientific Industries, Bohemia, NY, USA) every 10–15 min. The
residue must also be vigorously vortex-mixed after the superna-
tant is removed by centrifugation because the sediment can often
be difficult to separate and clumps interfere with visualization by
microscopy.
3.2. Numbers and types of diatoms extracted using rapid enzymatic
protocol
All 20 lung samples, including the periphery of the lungs into
which water would not readily penetrate post mortem, were
positive according to the rapid enzymatic protocol, and we
identified 60–23,000 diatom valves/g lung tissue (Table 3).
Representative freshwater diatoms belonging to the genera
Achnanthidium, Amphora, Asterionella, Cymbella, Diatoma, Eunotia,
Fragilaria, Gomphonema, Navicula, Nitzschia, Pinnularia, Surirella,
Ulnaria, Cyclotella, Melosira and others were found in cadavers 1–8.
Representative marine diatoms belonging to the genera Actino-
ptychus, Bacteriastrum, Biddulphia, Chaetoceros, Climacosphenia,
Diploneis (panduriform), Grammatophora, Licmophora, Rhizosolenia,
Campyloneis, Rhaphoneis, Thalassionema and others were also
identified in cadavers 9 and 10.
These results showed that rapid enzymatic digestion could
detect sufficient numbers and types of diatoms in small lung
samples during practical casework and suggested that all ten
victims had aspirated water before death. In particular, the
possibility of water aspiration was enhanced in eight victims
retrieved from shallow water (Cases 1, 2, 9, 10 from <30 cm of
 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185 183

Fig. 3. Microscopy image of residues and diatoms. (a) Comparison of residues prepared using both digestion methods. Quantity of final residue was appropriate and did not
interfere with observation by microscopy. (b) Image of diatoms extracted using rapid enzymatic digestion (high-power field).

water; Cases 4, 6, 7, 8 from < 90 cm) because water would not have
readily penetrated the peripheral regions of the lungs of these
victims after death.
The results of rapid enzymatic digestion agreed with all autopsy
findings (Table 2) including the conventional diatom test using
fuming nitric acid and the electrolyte findings of pleural effusions
(Supplementary Table S1 and S2). Given the possible uneven
distribution of diatoms throughout the lungs, the amount of lung
tissue (total 2 g from both lungs) might have been insufficient to
estimate the actual numbers of diatoms in whole lungs of victims.
This might be supplemented by analyzing several other parts of the
lungs. However, this would require more time, labor and cost.
184 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185

Table 3
Numbers of diatoms in cadavers determined using two digestive methods.

Case no. Rapid enzymatic digestion Conventional acid digestion (90–94% fuming nitric acid + 30% hydrogen peroxide)
(proteinase K + buffer ATL, 5N
hydrochloric acid)

Cadaver Cadaver Water

Right lung* Left lungy Right lung* Left lungz Kidney Liver Discovery site
(valves/g) (valves/g) (valves/g) (valves/g) (valves/10 g) (valves/10 g) (valves/mL)

1 240 130 380 190 0 0 77

2 4600 4400 7300 6600 2 1 120
3 220 210 470 300 0 0 660
4 300 320 500 570 1 1 340
5 1400 270 1700 570 0 0 420
6 8100 23000 3800 4200 0 0 380
7 7900z 2400 5400z 1400 0 0 750
8 2800 950 2500 2200 9 4 900
9 60 88 48 490 1 0 230
10 1100 890 700 880 0 1 13

Negative control did not contain diatoms.

*
Lower lobe of right lung (inner region).
y
Upper lobe of left lung (peripheral region).
z
Lower lobe of left lung in Case 7.

3.3. Advantages and limitations of rapid enzymatic digestion
3.3.1. Advantages
Diatoms can usually be extracted from two lung samples within
3 h using rapid enzymatic digestion, and thus samples can be
assessed by microscopy on the day of autopsy, which means that
the involved institutions can be informed of results much sooner
than before.
The presence of abundant diatoms in lung samples of a victim
would rapidly, and less laboriously than with the classical
diatom method, assists in the diagnosis of drowning when
characteristic macroscopic autopsy findings of drowning are
obvious and the environmental aspects and a police report agree
with the autopsy findings. Meanwhile, evidence that diatoms
are negligible or absent in lung samples would be helpful to
preliminary question, aspiration of liquid in victims retrieved
from aquatic environments, provided that the putative drown-
ing media contains a high concentration of diatoms. This would
also help to confirm that victims found on dry land actually
drowned when secondary delayed drowning or homicide
by drowning followed by disposal of the body on land is
suspected.
Conventional diatom testing using fuming nitric acid is
laborious and potentially dangerous. Therefore, this test can
sometimes be omitted when characteristic macroscopic autopsy
findings of drowning are obvious. However, some controversy
might arise if causes of death are determined only from autopsy
findings, toxicological test results, and environmental aspects
without considering positive findings of diatom tests. Therefore,
to examine at least the lungs would be appropriate when
characteristic macroscopic autopsy findings of drowning are
obvious and the probability of drowning is very high. Thus, rapid
enzymatic digestion could serve as a minimal test when
conventional diatom testing is not an option due to time or labor
constraints.
Rapid enzymatic digestion is simple and very effective for small
samples of lung tissue. Therefore, in selected cases, diatoms could
even be examined in lung samples obtained by biopsy without
autopsy.
Qiagen Proteinase K and Buffer ATL are available worldwide and
rapid enzymatic digestion is straightforward and reproducible. The
cost of the test can be covered in Japan by an allocated charge if the
amount of lung tissue is 1 g. Moreover, all manipulations and
reactions involved in the protocol can proceed in one 15-mL tube,
and the only instruments required are a low-speed centrifuge and a
block incubator (or water bath) if up to 1 g of lung tissue is
analyzed. The centrifuge speed should be the same as that in the
conventional diatom test because the specific gravity (1.03) of
Buffer ATL (lot no. 145038438) containing Qiagen Proteinase K (lot
no. 145046626) is lower than that of 90–94% fuming nitric acid
(1.50) and 60% nitric acid (1.38).
3.3.2. Limitations
Rapid enzymatic digestion is limited to lung tissues because
10–20 g of kidney or liver tissues would be required for sufficient
sensitivity, which would be too expensive and impractical for
routine testing. The positive rate of diatoms in closed organs or
bone marrow is not generally high (range: 58% in Ref. [16]; 46% in
Ref. [19]; 30% in Refs. [9,17]; 28% in Ref. [28]). Diatoms are difficult
to find in enclosed organs especially when their density in the
water where a victim was found is low. Even if diatoms are
detected in enclosed organs, providing information about aspirat-
ed water is difficult because the numbers and variations in types of
diatoms are low in such organs. A few diatoms can also be found
even in enclosed organs or the blood of non-drowned victims
[18,19,29]. On the other hand, diatoms found in heart, lung and
skin samples from experimental, submerged piglet carcasses have
provided evidence indicating post mortem contamination [30].
Moreover, a single diatom in muscle samples in that study
provided evidence of ante mortem contamination [30]. Using
conventional diatom testing, we found a 50% (5 of 10) ratio of
diatoms in enclosed organs and only one or two diatom valves per
10 g of kidney and/or liver samples except in those from putrefied
Case 8 (13 valves). Thus, a positive result cannot be expected from
1 g of closed organs (kidney and liver). Moreover, even 1 g of liver is
much more difficult to completely digest than lung tissues using
proteinase K and Buffer ATL.
If enclosed organs other than the lungs are needed for more
positive evidence of drowning because diatoms in enclosed organs
usually suggest that a victim had entered water while still alive,
then blood samples (cardiac and femoral blood) may be
substituted because the content of protein (and fat) is much
lower than that of the kidney and liver. Some investigators [29,31]
have described the extraction of diatoms from 5 or 6 mL blood.
Blood can be hemolyzed and solubilized by adding ultrapure water
and sodium dodecyl sulfate (SDS) and then most protein can be
 E. Kakizaki, N. Yukawa / Forensic Science International 251 (2015) 179–185
removed by centrifugation. When samples are processed in this
manner before enzymatic digestion, large volumes (10–20 mL) of
blood obtained at autopsy would be analyzed to increase
sensitivity.
Opinions about the diagnostic value of finding diatoms only in
the lungs vary. A cut-off value for the number of diatoms in lungs
might be required to estimate whether or not a victim aspirated
water before death. However, cut-off values would differ according
to the environment and to define them would require studies of an
enormous number of cases over a very long period. The diagnostic
value of diatom in the lungs should always be pondered together
with the state of preservation of the body and other autopsy
findings as well as with the water depth at which the body is
retrieved and other characteristic of the putative drowning media
such as its diatom concentration.
4. Conclusion
Rapid enzymatic digestion is proposed for detecting diatoms
during practical casework to assist with concluding whether or not
a victim died due to drowning. One gram of lung tissue was
effectively digested within 60 min using Qiagen Proteinase K and
Buffer ATL. The digestion process was completed by heating in 5N
hydrochloric acid, which dissolved tissue debris that had escaped
proteinase K digestion. The quantity of the final residue after
digestion was appropriate for visualizing diatoms by light
microscopy. Involved institutions can be informed of the results
of diatom test much sooner than before. This simple and rapid
procedure can be useful for assessing small lung samples in routine
medico-legal activities to help conclude drowning as a cause of
death.
Acknowledgements
We thank the staff at the Forensic Science Laboratory of
Miyazaki Prefectural Police Headquarters for the GC/MS and
alcohol analyses. We also thank Ms. Nahomi Takayama, Ms. Yuri
Inoue, Ms. Ai Sonoda and Ms. Fumina Shimoda for technical
assistance with the preliminary experiment, as well as Ms. Haruna
Matsuda for technical assistance with forensic autopsies. We
sincerely thank Dr. Tatsuo Shinohara, Shimizu Chuo Clinic in
Miyazaki, for his kind donation to support our research. This study
was supported by a Grant-in-Aid for Scientific Research (No.
25293163) from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.forsciint.2015.03.
025.
References
[1] P. Saukko, B. Knight, Knight’s Forensic Pathology, Chapter 16, Immersion Deaths,
third ed., Arnold, London, 2004, pp. 395–411.
[2] V.J. DiMaio, D. DiMaio, Forensic Pathology, Chapter 15, Death by Drowning,
second ed., CRC Press, Boca Raton, FL, 2001, pp. 399–407.
[3] P. Lunetta, J.H. Modell, Macroscopical, microscopical, and laboratory findings in
drowning victims, in: M. Tsokos (Ed.), Forensic Pathology Reviews, vol. 3,
Humana Press Inc., Totowa, NJ, 2005, pp. 3–77.
[4] M.H.A. Piette, E.A. De Letter, Drowning: still a difficult autopsy diagnosis, Forensic
Sci. Int. 163 (2006) 1–9.
185
[5] M.J. Shkrum, D.A. Ramsay, Forensic Pathology of Trauma: Common Problems for
the Pathologist, Chapter 5, Bodies Recovered from Water, Humana Press, Totowa,
NJ, 2007, pp. 243–293.
[6] E.J. Armstrong, Introduction, in: E.J. Armstrong, K.L. Erskine (Eds.), Water-Related
Death Investigation: Practical Methods and Forensic Applications, CRC Press, Boca
Raton, FL, 2011, pp. 1–25.
[7] N. Yukawa, E. Kakizaki, S. Kozawa, Chapter 1. Diatom and laboratory tests to
support a conclusion of death by drowning, in: G.N. Rutty (Ed.), Essentials of
Autopsy Practice: Innovations, Updates and Advances in Practice, Springer, Lon-
don, 2013, pp. 1–36.
[8] A. Auer, Qualitative diatom analysis as a tool to diagnose drowning, Am. J. Forensic
Med. Pathol. 12 (1991) 213–218.
[9] J.V. Pachar, J.M. Cameron, The diagnosis of drowning by quantitative and qualita-
tive diatom analysis, Med. Sci. Law 33 (1993) 291–299.
[10] B. Ludes, M. Coste, N. North, S. Doray, A. Tracqui, P. Kintz, Diatom analysis in
victim’s tissues as an indicator of the site of drowning, Int. J. Legal Med. 112 (1999)
163–166.
[11] J. Hürlimann, P. Feer, F. Elber, K. Niederberger, R. Dirnhofer, D. Wyler, Diatom
detection in the diagnosis of death by drowning, Int. J. Legal Med. 114 (2000)
6–14.
[12] E. Kakizaki, S. Kozawa, M. Sakai, N. Yukawa, Bioluminescent bacteria have
potential as a marker of drowning in seawater: two immersed cadavers retrieved
near estuaries, Legal Med. 11 (2009) 91–96.
[13] E. Kakizaki, S. Kozawa, H. Matsuda, E. Muraoka, T. Uchiyama, M. Sakai, N. Yukawa,
Freshwater bacterioplankton cultured from liver, kidney and lungs of a decom-
posed cadaver retrieved from a sandy seashore: possibility of drowning in a river
and then floating out to sea, Legal Med. 12 (2010) 195–199.
[14] E. Kakizaki, S. Kozawa, N. Imamura, T. Uchiyama, S. Nishida, M. Sakai, N. Yukawa,
Detection of marine and freshwater bacterioplankton in immersed victims:
post-mortem bacterial invasion does not readily occur, Forensic Sci. Int. 204
(2011) 80–87.
[15] E. Kakizaki, Y. Ogura, S. Kozawa, S. Nishida, T. Uchiyama, T. Hayashi, N. Yukawa,
Detection of diverse aquatic microbes in blood and organs of drowning victims:
first metagenomic approach using high-throughput 454-pyrosequencing, Foren-
sic Sci. Int. 220 (2012) 135–146.
[16] B. Ludes, S. Quantin, M. Coste, P. Mangin, Application of a simple enzymatic
digestion method for diatom detection in the diagnosis of drowning in putrefied
corpses by diatom analysis, Int. J. Legal Med. 107 (1994) 37–41.
[17] F. Bortolotti, G. Del Balzo, R. Calza, F. Valerio, F. Tagliaro, Testing the specificity of
the diatom test: search for false-positives, Med. Sci. Law 51 (2011) S7–S10.
[18] P. Lunetta, A. Miettinen, K. Spilling, A. Sajantila, False-positive diatom test: a real
challenge? A post-mortem study using standardized protocols, Legal Med. 15
(2013) 229–234.
[19] E. Kakizaki, K. Takahama, Y. Seo, S. Kozawa, M. Sakai, N. Yukawa, Marine bacteria
comprise a possible indicator of drowning in seawater, Forensic Sci. Int. 176
(2008) 236–247.
[20] H. Inoue, T. Ishida, A. Tsuji, K. Kudo, N. Ikeda, Electrolyte analysis in
pleural effusion as an indicator of the drowning medium, Legal Med. 7 (2005)
96–102.
[21] Y. Usumoto, N. Sameshima, W. Hikiji, A. Tsuji, K. Kudo, H. Inoue, N. Ikeda,
Electrolyte analysis of pleural effusion as an indicator of drowning in seawater
and freshwater, J. Forensic Legal Med. 16 (2009) 321–324.
[22] K. Matoba, M. Murakami, A. Hayakawa, K. Terazawa, Application of electrolyte
analysis of pleural effusion to diagnosis of drowning, Legal Med. 14 (2012) 134–
139.
[23] D. Yajima, H.K. Saito, K. Sato, M. Hayakawa, H. Iwase, Diagnosis of drowning by
summation of sodium, potassium and chloride ion levels in pleural effusion:
differentiating between freshwater and seawater drowning and application to
bathtub deaths, Forensic Sci. Int. 233 (2013) 167–173.
[24] J. Sambrook, D.W. Russell, third ed., Molecular Cloning, A Laboratory Manual,
Chapter 6, Preparation and Analysis of Eukaryotic Genomic DNA, vol. 1, Cold
Spring Harbor Laboratory Press, New York, 2001, pp. 6.1–6.12.
[25] W.M. Strauss, Preparation of genomic DNA from mammalian tissue, Curr. Protoc.
Mol. Biol. 42 (2001), 2.2.1-2.2.3.
[26] P.A. Dı́az-Palma, A. Alucema, G. Hayashida, N.I. Maidana, Development and
standardization of a microalgae test for determining deaths by drowning, Foren-
sic Sci. Int. 184 (2009) 37–41.
[27] A. DiGiancamillo, C. Domeneghini, D. Gibelli, C. Cattaneo, Diatom extraction with
HCl from animal tissues: a technical note, Legal Med. 13 (2011) 268–271.
[28] M.S. Pollanen, C. Cheung, D.A. Chiasson, The diagnostic value of the diatom test for
drowning, I. Utility: a retrospective analysis of 771 cases of drowning in Ontario,
Canada, J. Forensic Sci. 42 (1997) 281–285.
[29] Y. Seo, S. Sato, K. Kuroki, T. Kishida, A simple DNA coprecipitation method
for the detection of diatoms in heart blood, Forensic Sci. Int. 232 (2013)
154–159.
[30] A. DiGiancamillo, E. Giudici, S. Andreola, D. Porta, D. Gibelli, C. Domeneghini, M.
Grandi, C. Cattaneo, Immersion of piglet carcasses in water—the applicability of
microscopic analysis and limits of diatom testing on an animal model, Legal Med.
12 (2010) 13–18.
[31] N. Yoshioka, K. Takahashi, Demonstration of diatoms in heart blood, Res. Pract.
Forensic Med. 29 (1986) 57–61 (in Japanese with English abstract).