70% Ethylated and Non-Ethylated Swab: A Trace Evidence Recovery

Method and Usefulness in Spectacles Forensic Evidence

Simon Martin Manyanza Nzilibili

1,3

, Muh. Abduh Dwi Putra

, Ahmad Yudianto

1,2,4

Forensic Science Program, Post Graduate School, Airlangga University, 4-6 Airlangga Rd., 60286, Surabaya - Indonesia.

Forensics and Medico-legal Department, Faculty of Medicine, Airlangga University, Surabaya – Indonesia

Ministry of Health, Community Development, Gender Elderly and Children, Dodoma – Tanzania.

Human Genetic Laboratory, Institute of Tropical Disease, Airlangga University, Surabaya – Indonesia.

Keywords: Ethyl, Spectacle, Trace Evidence, 70% Ethylated swab

Abstract: Trace evidence recovery has been studied to involve a number of methods with the inclusion of swabbing

methods. In swabbing, different fluids have also been proposed with minimal highlight to ethylated swabbing

(prepared from ethanol). Through the use of swabbing fluid at 70% dilution (70% ethylated swab) run

simultaneously with distilled water swab, the findings discovered were that 70% ethylated swab was found to

concentrate large amount of DNA sample (1421µg/ml), twice the amount recovered by distilled water swab

(654.5µg/ml). In terms of purity of recovered DNA; 70% ethylated swab presented nearly similar purity with

distilled water DNA pellet (ethylated swab yielded 1.219 purity ratio while distilled water swab yielded

1.176). Electrophoresis DNA molecule migration displayed multiple bands in contrast with 70% ethylated

swab from positive to negative electrode. This shows the presence of both small and larger sized DNA

fragments. Distilled water swab displayed one deep band near negative terminal electrode. Thereby; the study

finding analysis illustrates and suggests that 70% ethylated swab is a useful and strong method in recovering

trace DNA evidences for successful DNA profile establishment. In addition, spectacle is prospective a

potential harbor of trace DNA sample for forensic investigation.

1 BACKGROUND

Recovery of micro- to macro-Deoxyribonucleic acid

(DNA) trace evidence is studied to involve adhesive

tape, forceps, and vacuum methods (Fisher et al.

2007; Ah Van Oorschot et al. 2010). Swab method is

also discussed and presented to work properly and is

far better, while moist in recovering such trace

evidences (van Oorschot et al. 2003; Ah Van

Oorschot et al. 2010; Jack Dillon, Debra Figarelli,

David Sylvester 2009; Oregon State Police 2015;

Puritan 2016; Adamowicz et al. 2014). It is opted and

used as moisturising fluid in case of remains of high

and efficacy determinant. To explore the

effectiveness, the experimental studies done have

explored the success of ethyl moist swabbing, though

most still recommend water moistening option (van

Oorschot et al. 2003; Raymond et al. 2008). Thus, in

the DNA retrieval study; 95% ethanol swab

(Slrichantrawonq et al. n.d.) was found to be suitable

to recover DNA at a high yield than distilled water

(Hildebrand et al. 2004), in which a 25% ethylated

alcohol swab yielded the most compared to those with

50% concentration and distilled water swab.

Emphasizing essence to these innovations is the

maximized retention of minute natured sample

residing on swab after recovery against loss or blow-

away (Fisher et al. 2007). Contrary to a noted good

use as swab fluid, ethyl on the other hand is presented

as a decontaminant and lyses catalyst (Gršković et al.

2013), which means that it has a destructive effect in

opposition to recovery usefulness. Despite of

substantial trials on alcohol fluid, specified ethyl

percentage which is effective to be used in swabbing

is yet to be established. This study therefore dedicated

furthered investigation on a 70% ethylated swab

supply on spectacle evidence convinced by reasons

below;

1.1 Why 70% Ethylated Alcohol Swab?

The percentage of alcohol on treatment of biological

sample has a direct relationship. On DNA extraction

and preservation usage, 70% ethyl has evidenced a

312

70 .

DOI: 10.5220/0007541903120316

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 312-316

ISBN: 978-989-758-348-3

flexible treatment of biological samples which allow

morphological exploration afterwards (Oswald

2007). This is in contrast to high or low percentage in

respect to a volatile, drying blow-away, non-flexible,

and degradation-prone performance. In addition;

compared to other alcohol groups such as

isopropanol, ethanol is highly precipitous that

resuspend DNA pellet easily. The insolubility of

DNA molecules is catalysed by forming H-bonds

with water during isolation (decrease hydration

ability of water to DNA) – reduced decaying. Lower

dielectric leads DNA to aggregate and concentrate

with cations below lighter molecules under phenol-

chloroform extraction (Brennan 2017; Zumbo 2013).

Referring to the percentage used in the reference

above, ethylated alcohol swab envision has a special

use in recovering DNA trace evidence. 70% ethyl

alcohol swab is optimised for consideration as

explained earlier. This ground prompted the

exploration of the usefulness of 70% ethylated swab

through admission of spectacle as useful and reliable

suggested source of trace biological evidence based

on Locard’s and Kick’s contact and silent witness

respective principles. Appreciation of 70% ethyl

swab and admission of spectacle, in addition to

normally referred evidences such as clothes, knife,

vehicles, firearms, bedding, food, condoms, lip

cosmetics, wallets, jewellery, glass, skin, bullet,

paper, cables, windows and door lockers/handle,

stones and watch (Ah Van Oorschot et al. 2010)

broaden exhibits. Either in recovery of such micro or

macro exhibits like hair, dust, soil, glass particles,

fluids, touched surfaces, clothes (Fisher et al. 2007)

as forensic evidence, limited information is on the

spectacles as potential source of biological trace

evidence. Apart from compilation through literature

reviews of the established useful properties of the

ethyl alcohol in forensic evidence; this study

complemented the findings used to recover trace

DNA from spectacles especially through the use of

70% saturation. Aggregation of these information

potentiate establishing special 70% ethyl swab for

trace evidences swabbing recovery as reported in this

study compared to most recommended water swabs.

2 MATERIALS AND METHODS

The article paralleled literature reviews on available

studies of ethylated alcohol swab application or

usefulness and experimental authentication of

spectacle evidence. Experimental content was

conducted at the University Human Genetic

Laboratory involving two biological samples

swabbed from two different spectacles: one by a 70%

ethyl swab and the other by a distilled water swab.

2.1 Sample Recovery

Samples to determine the usefulness and application

of ethylated swab was obtained from two participants

who voluntarily gave their spectacles after being

given a clear understanding of the study purpose.

From two spectacle evidences, DNA trace biological

sample was recovered by a separate swabbing under

one swabbing direction and surface without

repetition. The two swabs used were sterilised and

cotton-made. One swab was a readymade 70%

ethylated which swabbed one spectacle, and the other

was a dry swab which was moistened by 1cc of

distilled water and swabbed the second spectacle. The

process was immediately followed by a tube soaking

of spectacle swabbed swabs into 2 different tubes

filled by 4cc distilled water overnight to allow down

settling of DNA biological traces recovered for DNA

analysis.

2.2 DNA Extraction

The extraction process proceeded with removing of

upper most fluids while retaining down settled sample

solution. Then, 0.5cc of each sample was isolated in

a sterile centrifuge tube; pipetted with 1cc of DNAZol

(Invitrogen, ThermoFisher Scientific, Waltham, MA,

USA), and vortexed and incubated for 15 minutes.

Then, it was vortexed with 0.2cc of Chloroform

(Merck KGaA, 64271 Darmstadt, Germany)

followed with a centrifugation at 8,000 rpm for 10

minutes. Separated supernatant was obtained into

eppendorf with isopropanol 1cc (EMSURE®, Merck

KGaA, 64271 Darmstadt, Germany), 15 minutes

incubation, and centrifugation at 12,000 rpm for 10

minutes then with care followed by the discarding of

supernatant fluid again, leaving settled and

concentrated pellet. The pellet was washed with 0.5cc

of 70% ethanol (EMSURE®, Merck KGaA 64271

Darmstadt, Germany), and it then underwent 15

minutes of centrifugation at 12,000 rpm for 5

minutes, which again was followed by the removal of

supernatant through Chen et al. (2010) as well as

Chomczynski et al. (1997) protocols. Finally, 50µl of

distilled water resuspended formed DNA pellet for

spectrophotometer and electrophoresis.

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2.3 Spectrophotometer Measurements

Spectrophotometry measurement was done in order

to establish concentration and quality parameters of

70% ethylated swab in reference to distilled water

swab, as well as the potential extent of spectacle

evidence in yielding significant biological sample for

DNA. Using Ultraviolet-visible Spectrophotometer

(UV-1601, PC, Shimadzu, Japan), DNA

Concentration was determined by absorbance reading

at 260nm and 280 in UV-1061, while DNA Purity

was given by Optical Density (OD) OD260/OD280

ratio, refer Table 1.

2.4 Electrophoresis

Electrophoresis of acrylamide gel method was opted

due to sensitivity even to minute sample without

Polymerase Chain Reaction (PCR) (purposely to find

out if both 70% ethylated swab and spectacle

referenced to water swab can yield interesting results

without polymerase amplification). The gel was

prepared by a 3cc acrylamide reagent (Sigma-

Aldrich) mixed with 8cc Tris-borate-EDTA (TBE) -

0.5x (Promega Corporation, Madison, USA). Then,

Temed (Sigma-Aldrich) 20µl followed with 200µl

ammonium perisulfate solution (Sigma-Aldrich)

under homogenization cycles at 100 Volts for 60

minutes (Figure 3).

3 RESULTS AND DISCUSSION

3.1 70% Ethyl swab recovery method

According to literature, alcoholic swab has

exemplified usefulness in varied percentages. As

found from this study, the alcoholic swab with a

concentration of 70% was reasonably examined and

presented to substitute and establish a useful ethylated

swab potential for maximizing recovery of trace

biological evidence. Compared the two swabs used,

the 70% ethyl alcoholic swab and distilled water

swab, the findings presented closer results in

spectrophotometer measurement (Table 1) but quite

different in electrophoresis band contrast (Figure 3).

Concentration reading was measured almost three

times in 70% ethylated swab compared to distilled

water swab as per Figure 1. This concentration gives

the interpretation that 70% ethylated swab recovers

more DNA samples compared to possible amount

able to be recovered by distilled water swab. In

forensic profiling analysis, this interpretation gave a

meaning to the usefulness of increased probability

and assured the recovery of an adequate amount of

sample from targeted evidence of traces that is

potential to enable successful profiling results during

experimentation.

Table 1: Concentration and Purity of Spectacle DNA

evidence under ethylated and non-ethylated swab

Sample

Code

Absorbance

260 nm

Absorbance

280 nm

DNA

Concentration

(ng/ul)

DNA

Purity

Distilled

Water

Swab-A 0.187 0.159 654.5

1.17

70%

Ethylate

d Swab-

0.406 0.333 1421

1.21

In deducing the purity measurement, the

generated purity increased confidence for usage of

ethylated swab. Estimated chances of increased

degradation and destruction of genetic materials as

anticipated through previous few reported

applications in decontamination pose a contrary

scenario. According to the studies; ethyl being as

destructive agent forecasted expectation that this

study also significantly generated a lowered purity by

the fact of its destructive ability (micro-organisms

discussed similar to structure of traces). Purity of the

70% ethylated swab recovered sample was nearly

similar above distilled water swab. Despite the fact

that both 70% ethylated swab and distilled water

swab were below the recommended limits of purity

(1.6-2.0) (Table 1), the findings suggested that use of

modern extraction would purify to acceptable limits.

The nature of results measured project useful pellet

(of acceptable limit) with agreed contribution of

effective recovery of ethyl swab.

Figure 1: Concentration of DNA extracted from 70% ethyl

and distilled water swabbed spectacle.

DNA was recovered with ethyl swab concentrated

DNA amount more than twice as high (1421µg/ml) as those

concentrated by distilled water swab (654.5µg/ml). The

ethyl method concentrated DNA amount due to its ability

to recover even the stickiest sample traces as much as

possible.

Distilled

Water

Swab (A)

32%

Ethylated

Swab (B)

68%

DNA Concentration (ng/ul)

314

Figure 2: Purity of DNA extracted from 70% ethyl and

distilled water swabbed spectacle.

Purity was assessed and compared to sample extracted by

water swab/ 70% ethyl swab was measured to have pure

DNA in a similar ratio. Purity of DNA was well-detected

outside of quality range. Thus, this similar quality (though

a bit high in ethyl) complemented the significance of the

use of 70% ethylated swab.

3.2 Electrophoretic Reaction

Electrophoresis DNA band contrast appeared to both

swabs. Migration of DNA fragments from negatively

charged electrodes to positive was established in

different numerous band level especially to sample of

the 70% ethyl swab as presented in Figure 3. This

band contrast portrayed the size, length and strength

of the DNA extracted from these two kinds of swabs

(ethylated and water swabs). As discussed in

concentration and purity above (Table 1), DNA

obtained through 70% ethylated swab was with nearly

similar purity but higher collected amount as

portrayed in Figure 1. This information implis that

both swabs yielded pure DNA capable to be analyzed

in electrophoresis, as shown in Figure 2. The meaning

is that DNA has successfully been electrophorised as

full charged fragments and migrated to appropriate

contrast level. However, from the displayed bands,

ethylated swab contrasted more bands compared to

water swab. This suggests that 70% ethyl swab

recovered more amount of DNA sample with various

strength and size leading to a differed migration of

which small-sized fragments were lighter and

migrated faster with contrast level closer to Anode

electrode as referred in Figure 3. The longer and

larger sized DNA fragments recovered in ethylated

swab and water swab appeared to contrast closer to

cathode electrode due to slower migration of charged

fragments.

Figure 3: Electrophoresis band contrast of DNA sample

recovered through 70% ethyl and distilled water swabbed

spectacle evidences.

This interpretation suggests three things. Firstly,

both swabs (70% ethylated and distilled water)

recovered a significant amount, but ethylated swab

recovered more significant minute traces evidenced

by different band contrasts and even being

concentrated more at lower level (closer to positive

end). Secondly the purity of DNA sample recovered

by ethylated swab was higher with excess

concentration compared to water swab. Thirdly,

minute and increased extraction of sample was of

useful quality as being able to be profiled on

electrophoresis even without PCR primer

amplification.

3.3 Spectacle

As other evidences were found at crime scene through

this study, spectacle was evaluated to useful and

potential evidence able to be used as source of trace

DNA for profiling as a result of contact from humans

that used it before. Through a well-established

recovery method, spectacle exhibit can significantly

contribute to the logged and harbored amount of

DNA in contacted sample from specific individuals

used before.

4 CONCLUSION

DNA spectrophotometer and band visualization

contrast depicted and suggested 70% ethylated swab

Distilled

Water

Swab …

Ethylated

Swab (B)

51%

DNA Purity

315

to be a useful and strong method that recovers large

and enough samples for DNA profile establishment.

As for the reasons stated above in signifying

conduction of this study, 70% of ethyl is in the

manner recommended due to it being a flexible

percent that tolerates further morphological treatment

of DNA sample from recovery and let them in for a

temporal storage before processing in the laboratory.

The study has also brought attention to the

consideration of spectacle as potential source of DNA

sample either found at crime scene for criminal

linkages or to purposed forensic inquiry for

investigative profiling.

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