The Formation of Ethanol 

in Postmortem Tissues

Robert D. Johnson

Russell J. Lewis

Mike K. Angier

Nicole T. Vu

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

February 2004

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

 

Office of Aerospace Medicine

Washington, DC 20591

DOT/FAA/AM-04/4
NOTICE

This document is disseminated under the sponsorship of

the U.S. Department of Transportation in the interest of

information exchange. The United States Government

assumes no liability for the contents thereof.
i

Technical Report Documentation Page 

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. 

DOT/FAA/AM-04/4 

4. Title and Subtitle 5. Report Date 

The Formation of Ethanol in Postmortem Tissues February 2004 

7. Author(s) 6. Performing Organization Code 

Johnson RD, Lewis RJ, Angier MK, Vu NT 

9. Performing Organization Name and Address 8. Performing Organization Report No. 

FAA Civil Aerospace Medical Institute 

P.O. Box 25082 10. Work Unit No. (TRAIS) 

Oklahoma City, OK 73125 

12. Sponsoring Agency name and Address 11. Contract or Grant No. 

Office of Aerospace Medicine 

Federal Aviation Administration 13. Type of Report and Period Covered 

800 Independence Ave., S.W. 

Washington, DC 20591 

15. Supplemental Notes 

This work was accomplished under the approved task AM-B-02-TOX-204. 14. Sponsoring Agency Code 

16. Abstract 

During the investigation of aviation accidents, postmortem samples obtained from fatal accident victims are 

submitted to the FAA’s Civil Aerospace Medical Institute for toxicological analysis. During toxicological 

evaluations, ethanol analysis is performed on all cases. Many species of bacteria, yeast and fungi have the 

ability to produce ethanol and other volatile organic compounds in postmortem specimens. The potential for 

postmortem ethanol formation complicates the interpretation of ethanol-positive results from accident victims. 

Therefore, the prevention of ethanol formation at all steps following specimen collection is a priority. Sodium 

fluoride is the most commonly used preservative for postmortem specimens. Several studies have been 

published detailing the effectiveness of sodium fluoride for the prevention of ethanol formation in blood and 

urine specimens; however, our laboratory receives blood or urine in approximately 70% of cases. Thus, we 

frequently rely on tissue specimens for ethanol analysis. The postmortem tissue specimens received by our 

laboratory have generally been subjected to severe trauma and may have been exposed to numerous microbial 

species capable of ethanol production. With this in mind, we designed an experiment utilizing unadulterated 

tissue specimens obtained from aviation accident victims to determine the effectiveness of sodium fluoride at 

various storage temperatures for the prevention of microbial ethanol formation. We found that without 

preservative, specimens stored at 4°C for 96 h showed an average increase in ethanol concentration of 1470%. 

At 25°C, these same specimens showed an average ethanol increase of 1432% after 48 h. With the addition of 

1.00% sodium fluoride, there was no significant increase in ethanol concentration at either temperature.

17. Key Words 18. Distribution Statement 

Document is available to the public through 

the National Technical Information Service 

Postmortem Ethanol, Formation, Microbe, Aircraft 

Accident Investigation 

Springfield, Virginia 22161 

20. Security Classif. (of this reporte) 20. Security Classif. (of this page) 21. No. of Pages 22. Price 

Unclassified Unclassified 14

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized 

1

THE FORMATION OF ETHANOL IN POSTMORTEM TISSUES

INTRODUCTION

The Federal Aviation Administration’s (FAA) Civil 

Aerospace Medical Institute (CAMI) is responsible under 

Department of Transportation (DOT) orders 8020.11A 

and 1100.2C, to “conduct toxicologic analysis on specimens from ... aircraft accident fatalities” and “investigate 

... general aviation and air carrier accidents and search for 

biomedical and clinical causes of the accidents, including evidence of ... chemical (use).” Therefore, following 

an aviation accident, samples are collected at autopsy 

and sent to CAMI’s Bioaeronautical Sciences Research 

Laboratory, Forensic Toxicology Research Team where 

toxicological analysis is conducted on various postmortem 

fluids and tissues.

Ethanol analysis is one of the most common tests performed on forensic specimens. The presence of ethanol in 

aircraft accident victims constitutes an important part of 

both aircraft accident investigations and litigation. Using 

modern analytical techniques, there is little doubt as to the 

quantitative amount of ethanol present in a postmortem 

specimen. The origin of the ethanol detected, however, 

is an important variable that must be considered when 

interpreting ethanol results in postmortem specimens. 

The microbial formation of ethanol in postmortem 

specimens is the most significant problem encountered 

when evaluating ethanol results. The first report dealing 

with postmortem ethanol production in corpses appeared in 1936 (1). At that time, a postmortem increase 

in ethanol concentration was occasionally observed, but 

the source of the increase was unknown. The first studies to elucidate the mechanism of postmortem ethanol 

formation occurred in the early 1970s (2, 3). From 

these initial studies it became clear that microbes are 

responsible for postmortem formation of ethanol. Many 

species of bacteria, yeast and fungi produce ethanol and 

other volatile organic compounds as a byproduct of their 

metabolism (4-12). Candida albicans has been identified 

as the microbe most often responsible for postmortem 

production of ethanol in humans (9, 13). This species of 

yeast is commonly found in humans in vivo. However, 

microbes not normally found in vivo, such as the Mucor

species of mold, are commonly found in decaying organic 

matter and soil (14); they may be introduced from the 

environment if the circumstances surrounding death are 

violent, as is often seen in aviation accidents. After death, 

endogenous and/or exogenous microbes begin rapidly 

consuming glucose and other nutrients present in the 

body and produce ethanol and/or other organic volatiles as metabolic byproducts (12, 15-17). Under optimal 

conditions, substantial concentrations of ethanol may be 

formed within hours of death (18). The relatively short 

time required for microbes to begin producing ethanol 

complicates the interpretation of a positive ethanol result 

in postmortem specimens. The potential for postmortem 

ethanol formation is disconcerting when considering that 

a positive ethanol result may help discern the cause of an 

aviation accident. Therefore, the preservation of biological 

specimens to minimize postmortem ethanol formation 

has been a priority for nearly 30 years. 

The most commonly utilized substance for the preservation of postmortem specimens is sodium fluoride. The 

fluoride ion prevents the formation of polysaccharides 

by the microbe and, in turn, prevents microbial growth 

(19). Storage temperature and duration play an important 

role in the microbial formation of ethanol. Numerous 

studies have examined the amount of time required for 

the microbial production of ethanol and the optimum 

storage temperature for the prevention of postmortem 

ethanol in blood and urine specimens (18-20). Additionally, these studies have demonstrated the effectiveness of 

sodium fluoride for the prevention of microbial ethanol 

formation. There are, however, at least two published 

reports concluding that sodium fluoride may be ineffective for the prevention of ethanol formation in blood 

samples containing sufficiently high concentrations of 

C. albicans (2, 9).

The vast majority of research publications involving microbial ethanol formation investigate blood and/or urine 

spiked with glucose and C. albicans or other microbes. 

Blood is submitted to our laboratory in approximately 

70% of all cases, urine even less frequently. Thus, various 

tissues are often examined to evaluate ethanol concentration in aviation accident victims. Furthermore, the tissue 

specimens received by our laboratory typically have been 

subjected to trauma as a result of the violent nature of aviation accidents. As a result, the tissues may be contaminated 

with endogenous and/or exogenous microbes capable of 

producing significant amounts of ethanol. In a previous 

study using rDNA amplification, which examined 45 

blood and tissue specimens obtained from fatal aviation 

accident victims, 95% were contaminated with enteric 

bacteria capable of ethanol production (21). Therefore, 

we examined the formation of ethanol in unadulterated 
2 

tissue specimens obtained from fatal aviation accident 

victims, which were prepared both with and without 

sodium fluoride as a preservative and were stored for up 

to 96 h at either 4°C or 25°C.

MATERIALS AND METHODS

Chemicals and Solutions

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained using a 

Milli-QTplus Ultra-Pure Reagent Water System (Millipore®, Continental Water Systems, El Paso, TX). Sodium 

fluoride, t-butanol, acetaldehyde, methanol, 2-propanol, 

acetone, n-propanol, isobutanol, n-butanol, sec-butanol 

and ethanol were purchased from Sigma Chemical Company (St. Louis, MO) in high purity and used without 

further purification. Purchased ethanol controls were 

obtained as 25, 50, 100, 150 and 300 mg/dL ethanol 

standards from Restek Corporation (Restek Corp., 

Bellefonte, PA).

A volatiles calibration solution was prepared in a matrix 

of certified-negative human blood at a final concentration 

of 157 mg/dL ethanol, 33 mg/dL acetaldehyde, 158 mg/dL 

methanol, 157 mg/dL 2-propanol, 31 mg/dL acetone, 

241 mg/dL n-propanol, 32 mg/dL isobutanol, 80 mg/dL 

n-butanol and 80 mg/dL sec-butanol. Two mL portions 

of the calibrator solution were transferred to 16 x 100 

mm screw-cap culture tubes and stored at -20°C for up 

to 6 months.

The internal standard solution was prepared as 39 

mg/dL t-butanol and 0.40% sodium fluoride. Sodium 

fluoride was added to the internal standard solution to 

increase the ionic strength of this solution. The increased 

ionic strength of the aqueous phase causes a larger portion of the volatile organic compounds to move into the 

headspace of the vial. This technique is commonly used 

in volatile analysis and is known as “salting out” (22). 

The internal standard solution was stored at 4°C for up 

to 6 months. The sodium fluoride solution used in the 

homogenization procedure below was prepared weekly 

at a final concentration of 1.00% (w/v) and stored in the 

refrigerator at 4°C.

Instrumentation

Analyte separation was achieved using a Varian CP3800 gas chromatograph (GC) (Varian, INC., Palo Alto, 

CA) equipped with dual fused silica capillary columns 

obtained from Restek Corporation (Restek Corp., 

Bellefonte, PA). The columns were a Rtx-BAC1 (0.53 

mm i.d. x 3.0 μm x 30 m) and a Rtx-BAC2 (0.53 mm 

i.d. x 2.0 μm x 30 m). The GC was fitted with a Varian 

CombiPAL (Varian, INC., Palo Alto, CA) headspace 

GC injection system that allowed for automated sample 

pretreatment and injection. The CombiPAL was utilized 

in conjunction with a 1 mL SGE gas tight syringe (400 

psi max pressure) obtained from SGE (SGE Chromatography Products, Austin, TX). The sample volume was set 

to 500 μL. The specimens were incubated at 50°C for 

13.30 min prior to injection, and the syringe was heated 

to 50°C. The syringe fill speed was set to 100 μL/sec, and 

the injection speed was 150 μL/sec. Following sample 

injection, the syringe was flushed for 30 sec with carrier 

gas. Identification and quantitation of the analytes of 

interest was accomplished using a flame ionization detector (FID). Control of the GC system, integration of 

chromatographic peaks and communication with the GC 

system were achieved using a Dell Optiplex GX 1 (Dell, 

Round Rock, TX) personal computer system equipped 

with Varian Star Chromatography Workstation software 

version 5.3.1 (Varian, INC., Palo Alto, CA).

GC Method

The GC injector and detector temperatures were set 

at 150°C. The GC oven was held isothermally at 40°C 

for the duration of the 10-min run. Helium was used 

as the GC carrier gas at a flow rate of 20 mL/min. The 

gases required to operate the FID were hydrogen and 

compressed air and had flow rates of 30 and 300 mL/min, 

respectively. 

The Varian Star Chromatography Workstation software allowed for automated identification and integration 

of all obtained peaks. The software integrated all peaks 

in a chromatogram with a S/N of 5 or greater. Peak area 

was used for the quantitation of each analyte of interest 

from a chromatogram. The retention times for ethanol 

and other volatiles were established on both columns 

following the analysis of a prepared blood calibrator.

Analytical Parameters

Both the limit of detection (LOD) and the limit of 

quantitation (LOQ) were determined for the detection 

of ethanol using this technique. The LOD, as defined 

by our laboratory, is the lowest analyte concentration 

detected with a peak S/N of at least 5 and a retention 

time within ± 2% of a known standard. The LOD for this 

method was determined to be 0.10 mg/dL with respect to 

ethanol. The LOQ was the lowest concentration detected 

that met the parameters described above and had a S/N 

of at least 10. The LOQ for this method was determined 

to be 1.00 mg/dL with respect to ethanol. A representative chromatogram for the analysis of ethanol and other 

volatiles using this method can be seen in Figure 1.
3

Figure 1. A representative headspace GC chromatogram for the analysis of volatiles in a 

postmortem specimen. Peaks of interest are: 1. acetaldehyde, 2. methanol, 3. ethanol, 4. 

acetone, 5. isopropanol, 6. t-butanol, 7. n-propanol, 8. sec-butanol, 9. isobutanol and 10. nbutanol. 

1 2 3 4 5 6 7 8 9

Minutes

0

100

200

300mV

 1 

 2 

 3 

 4 

5

 6 

 7

 8

 9

 10

Specimen Selection and Preparation

Biological specimens obtained from fatal aircraft 

accident victims are stored at -20°C at CAMI for up to 

5 years following their initial analysis. Using our laboratory database, specimens were selected from cases where 

microbial contamination was suspected.

Tissue specimens were thawed, divided into two approximately equal parts and weighed. Specimens were 

diluted with 2 parts of either DDW or 1.00% sodium 

fluoride. The tissue samples were then homogenized using 

a PRO250 Post Mounted Homogenizer (PRO Scientific, 

Oxford, CT) employing a 10.0 mm saw-toothed generator set at 22,000 RPM. To prevent sample-to-sample 

contamination, the generator was thoroughly cleaned 

between tissue specimens. Following homogenization, 

both the water and 1.00% sodium fluoride tissue homogenates were divided again into two parts by transferring 

approximately half of the volume to clean 16 x 100 mm 

screw-capped culture tubes. All four samples from the 

original tissue specimen were labeled appropriately and 

placed on ice awaiting an initial volatile analysis.

Volatile Analysis

The instrument was calibrated using the previously 

described calibration solution. The calibration solution 

was run as a test mix each day prior to analysis to evaluate 

the operation of the instrument. Ethanol controls were 

run before, during and after sample analysis. Specimens 

were analyzed immediately after homogenization to establish a baseline concentration of volatiles. Following 

the initial analysis, each specimen was capped and placed 

either in a refrigerator at 4°C or on a shelf at 25°C. The 

homogenates remained either in the refrigerator or on 

the shelf for the remainder of the experiment. At various times following initial homogenization, a portion 

of each specimen was aliquoted into a headspace vial for 

volatile analysis.
4 

RESULTS

The specimens selected for this study had indications 

of microbial activity in the original volatiles analysis, as 

will be discussed in the next section. Identification of 

the microbial species was not attempted and is beyond 

the scope of this paper. Table 1 outlines the different 

specimen types chosen. Each specimen was periodically uncovered for sampling and, therefore, potentially 

exposed to the microorganisms ubiquitously present in 

the environment. However, specimens analyzed in this 

study were examined without the intentional addition 

of microbes or substrate.

The 9 tissue specimens selected for this study were each 

divided into 2 equal parts and homogenized after the addition of either water or 1.00% sodium fluoride. Volatile 

analysis was immediately performed on these samples. 

Following specimen homogenization, both water and 

sodium fluoride homogenates were again split, and the 

specimens were then stored at either 4°C or 25°C. The 

samples stored at 4°C were re-examined for ethanol and 

other volatiles at t = 24, 48, 72 and 96 h. The samples 

stored at 25°C were re-examined for ethanol and other 

volatiles at t = 24 and 48 h.

An initial comparison of the 9 specimens homogenized in water and 1.00% sodium fluoride yielded the 

following results. The range and average initial ethanol 

concentration (t = 0) for these specimens was the same 

for both groups and was 1 - 28 mg/hg and 11 mg/hg, 

respectively. Table 1 shows the individual initial ethanol 

concentrations present in these samples.

The samples homogenized in 1.00% sodium fluoride 

and stored at 4°C and 25°C demonstrated no significant 

difference in ethanol concentrations between the two 

storage temperatures throughout the duration of the 

experiment. The largest individual increase in ethanol 

concentration observed for the sodium fluoride group 

occurred in a kidney specimen (#7) stored at 4°C. This 

specimen had an initial ethanol concentration of 28 

mg/hg and at 96 h contained 33 mg/hg ethanol. This 

slight increase, however, was likely due to experimental 

and/or instrumental error. The initial and final ethanol 

concentrations for all sodium fluoride preserved specimens 

can be seen in Table 2.

The 9 tissue specimens homogenized without preservative and subsequently stored at 4°C demonstrated 

significant ethanol formation over the course of the 

experiment. The average ethanol increase for these 9 

specimens stored at 4°C was 1470% after 96 h, with 

increases ranging from 107% to 7500%. The largest 

individual increase in ethanol concentration observed 

occurred in a kidney specimen (#8). While it initially 

contained 1 mg/hg ethanol, at 96 h this specimen was 

found to contain 76 mg/hg ethanol. This specimen had a 

net ethanol concentration increase of 75 mg/hg over the 

course of the experiment, an increase of 7500% over its 

initial ethanol concentration. The initial and final ethanol 

concentrations for the specimens investigated without 

preservative and stored at 4°C are shown in Table 3. 

The non-preserved specimens were also examined at 

25°C after 24 and 48 h. As expected, all 9 showed a 

dramatic increase in ethanol concentration. The average 

Table 1. Specimen types and initial ethanol concentrations. 

Specimen Number Specimen Type Ethanol Concentration 

(mg/hg) 

1 Muscle 6 

2 Kidney 18 

3 Muscle 4 

4 Kidney 4 

5 Muscle 23 

6 Kidney 5 

7 Kidney 28 

8 Kidney 1 

9 Muscle 2 
5

increase in ethanol concentration at 25°C for these 9 

specimens was 1432% at 48 h, with increases ranging 

from 109% to 6100%. The largest individual increase 

in ethanol concentration observed occurred in a kidney 

specimen (#8), with an initial ethanol of 1 mg/hg and 

an ethanol of 62 mg/hg at 48 h. The individual ethanol 

concentrations, at t = 0 and 48 h, for the specimens stored 

without preservative at 25°C are shown in Table 3.

DISCUSSION

The main objective of this study was to compare 

postmortem ethanol formation in unadulterated tissue 

specimens homogenized in either water or 1.00% sodium 

fluoride and stored at both 4°C and 25°C. We examined 

a single tissue specimen from each of 9 separate aviation 

fatalities for the formation of postmortem ethanol. All 

cases selected were chosen because they were suspected 

of microbial contamination. This suspicion arose due 

to either an abnormal distribution of ethanol in various 

tissues and fluids initially examined or abnormally high 

concentrations of various volatile organic compounds not 

normally present in vivo. As has been well documented, 

under normal circumstances following ingestion, ethanol 

distributes throughout the body according to the water 

content of various tissues and fluids (23-28). Therefore, 

when abnormal ethanol distribution is observed between 

two or more tissues and/or fluids from one victim, postmortem ethanol formation may be suspected. Another 

commonly used criteria to evaluate postmortem ethanol 

formation is the presence of other volatiles in a specimen. 

Volatiles such as acetaldehyde, n-propanol, sec-butanol, 

isopropanol, acetone and iso-butanol, when discovered 

during routine ethanol analysis, suggest the possibility of 

microbial formation of ethanol (4, 10, 13, 18, 29-32).

One of the initial goals of this study was to make 

a direct comparison between the increase in ethanol 

concentration in specimens homogenized in water and 

1.00% sodium fluoride. The differences observed between these two groups were substantial. Out of the 36 

individual experiments performed, i.e., 18 in water at 

4°C or 25°C and 18 in 1.00% sodium fluoride at 4°C 

or 25°C, all 18 of the non-preserved samples showed a 

significant increase in ethanol concentration between the 

 2

Table 2. The effect of temperature on ethanol production in specimens homogenized in 

1.00% sodium fluoride. 

Specimen # Preservative Temperature Initial Ethanol 

(mg/hg) 

Final Ethanol*

(mg/hg) 

4°C 6 1 NaF 

25°C

6 6

4°C 20 2 NaF 

25°C

18 16

4°C 3 3 NaF 

25°C

4 3

4°C 2 4 NaF 

25°C

4 4

4°C 23 5 NaF 

25°C

23

22

4°C 5 6 NaF 

25°C

5

6

4°C 33 7 NaF 

25°C

28 29

4°C 1 8 NaF 

25°C

1 1

4°C 1 9 NaF 

25°C

2 1

* Final ethanol concentrations were recorded after an incubation time of 48 h at 25°C and 96 h at 4°C.
6 

initial and the final measurement. While both the 4°C 

and 25°C groups without preservative showed dramatic 

increases in ethanol concentration, the increase occurred 

at a slower rate in the refrigerated specimens. Conversely, 

the 18 specimens preserved with 1.00% sodium fluoride 

demonstrated insignificant increases in ethanol concentration. These data are best represented graphically (Fig. 

2 - 10), where ethanol concentration is plotted versus 

time for all 9 specimens examined. Each Figure displays 

the results of the four experiments performed on each 

specimen. After completion of this cursory comparison, 

it was clear that sodium fluoride was extremely effective 

at inhibiting microbial ethanol formation. This agrees 

well with other sodium fluoride-preservative publications (18-20). 

In a majority of the specimens investigated, the overall increase in ethanol concentration after 96 h at 4°C 

was similar to the increase observed after 48 h at 25°C. 

This comparable concentration of ethanol formation 

was expected and may be explained by examining three 

factors. First, at lower temperatures, one would expect 

the metabolic rate of microbes responsible for ethanol 

formation to be slower than those at room temperature. 

Second, the formation of acids from the fermentation of 

carbohydrates, amino acids and alcohols may result in 

a pH that is harmful to pH-sensitive organisms. Lastly, 

there is a finite amount of substrate available in these 

specimens for the microbes to consume, and after the 

substrate supply has been depleted, no further ethanol 

can be produced. Therefore, the time required to consume all available substrate and/or create an unsuitable 

environment should take longer at lower temperatures, 

but the amount of ethanol produced as a byproduct of 

this metabolism should be similar. 

Six of the specimens investigated were initially reported 

negative for ethanol, as defined by our laboratory’s criteria (< 0.010%, < 10 mg/hg, < 10 mg/dL). After 96 h 

of storage at 4°C with no preservative, all 6 specimens 

had ethanol concentrations above our laboratory’s cutoff. 

Furthermore, 5 of the 6 were above 0.040% (40 mg/hg, 

40 mg/dL), which is the FAA’s legal cutoff for ethanol 

(14CFR91.17). At 25°C, all 6 previously reported negative specimens had values above 0.010% after 48 h, with 

4 of the 6 above 0.040%.

The results of this study reveal several trends. First, at 

both temperatures, ethanol was formed in all of the specimens when sodium fluoride was absent. Second, ethanol 

formation rate was slower at reduced temperatures, but 3

Table 3. The effect of temperature on ethanol production in specimens homogenized in water. 

Specimen # Preservative Temperature Initial Ethanol 

(mg/hg) 

Final Ethanol*

(mg/hg) 

% Increase 

4°C 28 367 1 None 

25°C

6 25 317

4°C 45 150 2 None 

25°C

18 40 122

4°C 46 1050 3 None 

25°C

4 30 650

4°C 40 900 4 None 

25°C

4 88 2100

4°C 80 252 5 None 

25°C

23

48 109

4°C 49 880 6 None 

25°C

5

71 1320

4°C 58 107 7 None 

25°C

28 90 221

4°C 76 7500 8 None 

25°C

1 62 6100

4°C 43 2050 9 None 

25°C

2 41 1950

*Final ethanol concentrations were recorded after an incubation time of 48 h at 25°C and 96 h at 4°C.
7

0

5

10

15

20

25

30

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 1, NaF @ 4C

Specimen 1, water @ 4C

Specimen 1, NaF @ 25C

Specimen 1, water @ 25C

Figure 2. Ethanol concentration vs. time for specimen # 1.

0

10

20

30

40

50

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 2, NaF @ 4C

Specimen 2, water @ 4C

Specimen 2, NaF @ 25C

Specimen 2, water @ 25C

Figure 3. Ethanol concentration vs. time for specimen # 2. 

0

10

20

30

40

50

60

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 3, NaF @ 4C

Specimen 3, water @ 4C

Specimen 3, NaF @ 25C

Specimen 3, water @ 25C

Figure 4. Ethanol concentration vs. time for specimen # 3. 
8 

0

40

80

120

160

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 4, NaF @ 4C

Specimen 4, water @ 4C

Specimen 4, NaF @ 25C

Specimen 4, water @ 25C

Figure 5. Ethanol concentration vs. time for specimen # 4. 

0

20

40

60

80

100

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 5, NaF @ 4C

Specimen 5,water @ 4C

Specimen 5, NaF @ 25C

Specimen 5,water @ 25C

Figure 6. Ethanol concentration vs. time for specimen # 5. 

0

20

40

60

80

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 6, NaF @ 4C

Specimen 6, water @ 4C

Specimen 6, NaF @ 25C

Specimen 6, water @ 25C

Figure 7. Ethanol concentration vs. time for specimen # 6. 
9

0

20

40

60

80

100

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 7, NaF @ 4C

Specimen 7, water @ 4C

Specimen 7, NaF @ 25C

Specimen 7, water @ 25C

Figure 8. Ethanol concentration vs. time for specimen # 7.

0

20

40

60

80

100

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 8, NaF @ 4C

Specimen 8, water @ 4C

Specimen 8, NaF @ 25C

Specimen 8, water @ 25C

Figure 9. Ethanol concentration vs. time for specimen # 8. 

0

10

20

30

40

50

0 24 48 72 96

Time

Ethanol Concentration

(mg/hg)

Specimen 9, NaF @ 4C

Specimen 9, water @ 4C

Specimen 9, NaF @ 25C

Specimen 9, water @ 25C

Figure 10. Ethanol concentration vs. time for specimen # 9. 
10 

with time, the refrigerated specimens produced ethanol 

concentrations similar to those at room temperature. 

Third, ethanol formation was virtually eliminated when 

specimens were homogenized in 1.00% sodium fluoride 

and stored at either 4°C or 25°C.

During the course of our experiments, we observed 

various volatile organic compounds being formed in 

these samples. We also observed volatiles, which were 

initially present, decrease in concentration over time. 

Some examples of volatiles that were either present 

initially or formed with time are acetaldehyde, n-propanol, sec-butanol, isopropanol, acetone and iso-butanol. 

These compounds, if present, were quantitated for each 

specimen at each analysis time, but after collection of 

the data, and subsequent data analysis, no discernable 

pattern could be established. 

CONCLUSIONS

It is clear from these experiments that all 9 unadulterated specimens from actual aviation accident victims 

contained microbes capable of ethanol production. We 

have demonstrated that even at 4°C, significant amounts 

of postmortem ethanol can form in the absence of a 

preservative. Additionally, we have demonstrated that 

the addition of sodium fluoride to postmortem tissue 

specimens during the homogenization process prevents the 

formation of ethanol at storage temperatures of 4°C and 

25°C. Therefore, we believe that sodium fluoride should 

be added as a precaution to all postmortem specimens. 

However, caution must always be used when interpreting 

ethanol results from postmortem samples since, even with 

these precautions, we cannot rule out the phenomenon of 

ethanol production in a small percentage of cases where 

larger numbers of microbes are present when the sample 

is received.

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