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Graphical Abstract

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Time evolution of electrical impedance spectra of gram-positive
and gram-negative bacteria
David Alejandro Miranda Mercado, Erika Viviana Godoy Alarcon, Ely Dan-
nier Valbuena Niño

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Highlights

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Time evolution of electrical impedance spectra of gram-positive
and gram-negative bacteria
David Alejandro Miranda Mercado, Erika Viviana Godoy Alarcon, Ely Dan-
nier Valbuena Niño

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• The time evolution of the electrical properties of Gram-positive and
Gram-negative bacteria in aqueous suspensions with methyl violet and
Lugol were analyzed.

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• The time evolution, which is associated with bacteria growth, was ana-
lyzed by linear regression of the electrical impedance for each excitation
frequency.
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• The time evolution of the electrical impedance spectra can be used to
differentiate Staphylococcus aureus from Escherichia coli bacteria.
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Time evolution of electrical impedance spectra of

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gram-positive and gram-negative bacteria
David Alejandro Miranda Mercadoa , Erika Viviana Godoy Alarconb , Ely
Dannier Valbuena Niñoa
a
Universidad Industrial de Santander, Cra 27 Cll

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9, Bucaramanga, 680002, Santander, Colombia
b
Institute of Chemistry, São Paulo State University (UNESP), CP
355, Araraquara, 14800-060, São Paulo, Brazil

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Abstract

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This research work reported the time evolution of the electrical properties
of gram-positive and gram-negative bacteria in aqueous suspensions with
methyl violet and Lugol. Measurements of galvanostatic electrical impedance
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spectra were performed in a frequency range from 10 Hz to 100 kHz. The
impedance magnitude as a function of frequency for methicillin-resistant
Staphylococcus aureus (gram-positive) and Escherichia coli O157:H7 (gram-
negative) strains in the presence of methyl violet, and Lugol showed that
both strains exhibited a progressive decrease in electrical impedance mag-
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nitude with increasing bacterial population, but the variation of impedance

magnitude rate in time is completely different between gram-positive and
gram-negative strain. The results suggest that the time evolution of elec-
trical impedance spectra can be used to differentiate Staphylococcus aureus
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from Escherichia coli bacteria.

Keywords:
Escherichia coli, Impedance measurement, Lugol, Methyl violet,
Staphylococcus aureus
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1. Introduction
Bacteria are cellular biological organisms that can be classified into two
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types, gram-positive and gram-negative, based on the interaction between

Email address: dalemir@uis.edu.co (David Alejandro Miranda Mercado)

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Preprint submitted to Elsevier September 2, 2022

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 ed
their cell walls and the gram stain components (see Fig. 1), i.e., methyl violet
and Lugol (iodine/iodide). Several layers of peptidoglycans (15 nm−80 nm)

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and lipoteichoic acids are the mean components of the cell wall in gram-
positive bacteria (see Fig. 1(a)). In comparison, cell walls in gram-negative
bacteria are constituted by a thin layer of peptidoglycan (10 nm) surrounded
by an external surface composed of lipopolysaccharide (see Fig. 1(b)) [1].

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(a) (b)

Figure 1: Schematic exemplification of a bacterial medium in a four-electrode cell contain-

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ing two types of bacteria with different wall structures: (a) Gram-positive and (b) gram-
negative bacteria exposed to Gram stain reagents, violet crystals, and Lugol dissolved in
PBS and TSB. The interaction of the crystal violet-Lugol pair with the wall bacteria and
its electrical response will depend on the chemical nature of the wall bacteria.
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Most antibiotics exhibit activity affecting the bacterial cell wall [2, 3];
thus, the unknowledge of wall type makes it difficult to appropriately choose
antibiotics and contributes to the actual growing bacterial resistance problem
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[4, 5]. The Gram stain method is used in the initial stage of bacterial diagno-
sis because it can provide early information for a timely diagnosis of infection
and orientate initial antibiotic treatment before culture results are available
[6]. The traditional Gram staining procedure consists mainly of five stages:
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(i) heat-fixed bacteria are stained with methyl violet, (ii) a mordant, a solu-
tion of iodine and potassium iodide, is added to bacterial samples, (iii) the
decolorizer is applied to remove the stain, (iv) a second stain is incorporated,
and finally, (v) stained bacteria are examined under optical microscopy [7].
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This procedure takes between 5 and 10 minutes to perform in the laboratory,
and the successful results depend on trained operators and their skills, ex-

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pertise, and experiences. Although Gram staining has been used for several
years, it would be convenient to develop an automated method for bacterial
differentiation that has low operation costs, a short detection time, and no
need for an expert to apply the method.
The culture medium supporting the growth of microorganisms changes its

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chemical composition with time, as nutrients are consumed and metabolized;
these changes are associated with a decrease in electrical impedance with the
bacterial growth time [8]. The electrical impedance is the opposition of a ma-
terial to the flow of a sinusoidal alternating current (AC) in a frequency range

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[9]. The electrical spectroscopy impedance (EIS) technique used in biological
assays to detect microbial growth is known as impedance microbiology; this
technique is based on analyzing changes in electrical impedance in culture

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media that are affected by microbial metabolism, mainly by the release of
ionic metabolites during bacterial growth [10, 11, 12, 13, 14, 15, 16, 17].
In classic impedance microbiology, a pair of electrodes submerged in a
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culture medium allows the measurement to be performed; this provides a
rapid and successful method of automation that is the basis of several com-
mercial analytical instruments for monitoring microorganisms, such as the
Bactometer (Bio Merieux, Nuertingen, Germany), Malthus systems (Malthus
Instruments Ltd., Crawley, UK), the rapid automated bacterial impedance
technique (RABIT) (Don Whitley Scientific Ltd., Shipley, UK), and Bac-
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Trac (Sy-Lab, Purkersdorf, Austria) [10, 11, 12, 13, 14, 18, 19].
This research work evaluated the time evolution of the electrical impedance
spectra (EIS) of gram-positive and gram-negative bacteria in a liquid medium
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using the main Gram components, methyl violet, and Lugol. Consequently,
we found qualitative differences in electrical properties between Escherichia
coli O157:H7 (gram-negative) and Staphylococcus aureus (gram-positive).
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2. Materials and methods

The method and materials used in measurements of the time evolution of
electrical properties of gram-positive and gram-negative bacteria in aqueous
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suspension with methyl violet and Lugol are reported below.

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2.1. Electrode surface cleaning

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Prior to the electrical impedance measurements, the electrode surfaces
were cleaned chemically and electrochemically by applying several consec-
utive voltammetric oxidation-reduction cycles; in this sense, four wires 24
of karat gold (1.0 mm in diameter and 1.0 cm in length) were cleaned with
Piranha solution (a 1 : 3 mixture of 30%v/v H2 O2 / concentrated H2 SO4 ) for
10 minutes and then rinsed with water (type I). Additionally, electrodes were

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electrochemically cleaned by cyclic voltammetry in 0.5 molar (0.5 M ) sulfu-
ric acid (H2 SO4 ) solution (20 cycles from −1.0 V to 1.6 V versus Ag/AgCl
(3.0 M KCl) at a scan rate 10 mV /s), until obtaining the characteristic be-
havior of a gold (Au) electrode (see Appendix A) [20, 21, 22, 23, 24, 25].

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2.2. Chemical solutions
The solutions were prepared with deionized water (18 M Ωcm) and ana-
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lytical grade chemicals. Sterile phosphate-buffered saline (PBS) at a concen-
tration of 10X) was prepared with 1.38 mM NaCl, 30.0 mM KCl, 81 mM
Na2 HPO4 , and 15 mM KH2 PO4 , and the pH was adjusted at 7.4 with NaOH;
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likewise, PBS at a concentration of 1X was prepared to dilute stock solution
PBS 10X 1 : 10 with deionized water. Serial dilutions were prepared from
freshly prepared methyl violet solution (10 g/L water) by adding PBS 1X
until 1.6 × 105 g/L methyl violet solution was achieved [26]. Moreover, a
commercial Lugol solution (1 : 5 KI/I2 ) was used.
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Bacteriological agar (Oxoid) and brain heart infusion (BHI), pH 7.4‡0.2

at 25 ◦ C, Merck, were used to prepare the solid culture; the liquid medium
was tryptic soy broth (TSB), pH 7.3‡0.2, Bacto; both media were prepared
according to the manufacturers instructions and autoclaved at 121 ◦ C and
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14.5 atm for 15 minutes.

2.3. Bacterial culture

The strains of Escherichia coli O157:H7 and methicillin-resistant Staphy-
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lococcus aureus (MRSA) were our models of gram-positive and gram-negative

organisms, respectively. Both bacteria were grown on agar BHI in Petri
dishes at 37 ◦ C for 24 h, from which an inoculum was taken and incubated
in 3 mL of TSB at 37 ◦ C and 200 rpm for 12 h. This culture was diluted
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at 1 : 10, 000 in TSB to obtain 1x103 CF U/mL − 1x104 CF U/mL. Finally,

100 µL from this culture was added to 96-well plates with 100 µL TSB.
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2.4. Electrical impedance spectroscopy measurements
Impedance measurements were carried out in galvanostatic mode using

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a Methrom Autolab PGSTAT204 potentiostat/galvanostat controlled by the
NOVA program. Impedance measurements were performed at frequencies fn
from 100 kHz to 10 Hz (where n = 1, 2, · · ·, 80) with a current amplitude
of 10 µA; the experimental setup consisted of a four-electrode cylindrical
cell designed according to the general configuration described by [27] (see

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Fig. 2); to determine the most appropriate measurement current based on
previously published results, electrical impedance measurements were per-
formed at currents of 0.1 µA, 1.0 µA, 10.0 µA, and 40.0 µA (see Appendix
B) [27, 28].

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Figure 2: Experimental setup for electrical impedance spectroscopy measurements: (a)

The setup implemented in the laboratory,(b) the four-electrode cell with dimensions of
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10 mm deep × 10 mm diameter in the center hole and with a capacity of 300 µL has
four equidistant cylindrical holes where the gold electrodes (99.9%, 10 mm long × 10 mm
diameter) are fixed. According to the scheme (c), in the four electrode setup, the signal
current passages across WE and CE, while the resulting potential is the measurement
between RE and S.
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Electrical measurements were made in triplicate and divided into three

stages for each strain. In the first stage, 40 µL of cultured bacteria were
added to the measuring cell with 200 µL of PBS. In the second stage, 40 µL
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of cultured bacteria and 20 µL of methyl violet solution (the methyl violet
concentration was chosen as presented in the Appendix C) were added to

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the measuring cell with 180 µL of PBS. In the third stage, 40 µL of culture
bacteria, 20 µL of methyl violet solution, and 20 µL of Lugol were added
to the measuring cell with 160 µL of PBS. In each stage, the measurements
were carried out for three hours at the initial time (t1 , 1 × 103 CF U /mL −
1 × 104 CF U /mL). Considering the low value of CFU at that time, it was

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not possible to use techniques such as enzyme-linked immunosorbent assay
(ELISA) to analyze the bacterial counts. Therefore, it was necessary to
evaluate it by the plate count method: a sample of the culture at each time
of electrical measurement was incubated for 16 h at 37 ◦ C in an agar plate

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and the number of colonies were counted [29].

2.5. Time evolution data treatment method

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The time evolution, which is associated with bacteria growth, was an-
alyzed by linear regression of electrical impedance for each excitation fre-
quency, as described below. First, the electrical impedance spectra Z(ω (n) ; tm )
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measured at an angular frequency ω (n) = 2πfn (with fn in the range described
above) at different times tm were used to obtain the impedance magnitude
(n) (n)
Zm = |Z(ω (n) ; tm )|, conforming to a data set {tm , Zm }. Second, the ratio
of impedance magnitude in time dZ/dt and the linear correlation coefficient
(n)
LCC were obtained by linear regression of the set {tm , Zm }; here, dZ/dt
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is the slope obtained in the linear regression and LCC, its linear correla-
tion coefficient. Third, the first and second steps are repeated at the other
frequencies fn . Finally, a plot of dZ/dt versus frequency is obtained.
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3. Results and discussion

The results and discussion of the measurements of electrical impedance
performed with the Staphylococcus aureus and Escherichia coli bacteria are
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reported below.

3.1. Effects of TSB, methyl violet, and Lugol on the electrical properties of
PBS
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We evaluated, in three steps, the effects on electrical properties due to

the addition of TSB, methyl violet, and Lugol to PBS before introducing
bacterial cells (see I to III in Fig 3). The first step (I) corresponds with PBS
+ TSB. In Fig 3, the magnitude of the electrical conductivity σ was obtained
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by Eq. (1), where σ(ω) depends on the angular frequency ω = 2πf of the
disturbance signal used for the electrical impedance measurement Z(ω) of

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the electrolyte solution, and h = 10−3 m corresponds to the height of the
liquid in the cylindrical cell [27].

ln(2) 1
σ(ω) = (1)
πh Z(ω)

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We observed that the electrical conductivity of PBS, (0) as shown in
Fig 3, is higher than that of PBS + TSB, (I) in Fig 3. PBS has strong elec-
trolytes, including Na+ , K+ PO3−
4 and Cl , which contribute to the electrical
−

conductivity. In the first setup, the addition of TSB decreases the electri-

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cal conductivity of PBS, Fig 3; this phenomenon is observed in the entire
frequency range.

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Figure 3: Electrical impedance magnitude (orange) and conductivity magnitude (brown)

of sterile solutions, both as a function of frequency for (0) PBS, (I) PBS + TSB, (II) PBS
+ TSB + methyl violet and (III) PBS + TSB + methyl violet + Lugol.
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The second step (II) consisted of the addition of methyl violet to the first
step. The increase, compared with PBS alone, in the electrical impedance
magnitude in Fig 3 for methyl violet dilutions is observed as a decrease
in the electrical conductivity on (II) in Fig 3. Despite the methyl violet
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concentration selected for our experiments, i.e., 4.0×10−5 mM , was the lowest
concentration used to introduce a few variations in the electrical properties
of PBS. This impedance at higher frequencies (f > 103 Hz) increased nearly
50% with respect to PBS due to the presence of TSB and methyl violet in
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PBS; however, at low frequencies (f < 102 Hz), the increase was lower than
5%.

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The third step (III) was achieved by adding Lugol to the second step.
The electrical conductivity, at the lower frequency, between the second and
third steps increases near 20%, and the impedance amplitude decreases by
approximately 25%.

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3.2. Cell cultures and electrical impedance spectra
Gram-positive (Staphylococcus aureus) and gram-negative (Escherichia
coli ) cell cultures were prepared as described in the Materials and Meth-
ods. Their electrical properties were analyzed by electrical impedance spec-
troscopy (Fig 4) by suspending colony-forming units (CFU) in a PBS so-

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lution with methyl violet and TSB for each of the incubation times 0 h,
1 h, 2 h and 3 h. The electrical behavior (Fig 4) can be associated with

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the increase of metabolites of ionic nature, such as K+ and Na+ , which are
exchanged through the bacterial wall with the external medium, during bac-
terial growth (see Appendix D). The growth curves exhibit exponential
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behavior, increasing from 104 CF U/mL to 106 CF U/mL in 3 h of incuba-
tion, where Staphylococcus aureus grows at a faster rate than Escherichia
coli.
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(a) (b)

Figure 4: Bode diagrams of the impedance magnitude of (a) Staphylococcus aureus (gram-
positive) and (b) Escherichia coli (gram-negative) in PBS, TSB and methyl violet (without
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Lugol) suspension at different incubation times.

At incubation time zero (t = 0 h) and at low frequencies (f < 100 Hz),

the highest impedance values are observed for the two strains (Fig 4) due
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to the amount of ionic metabolites in the electrolyte solution, typical of the
catabolism of the bacteria at the beginning of the measurement, and due

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to the lower bacterial population compared to those of longer incubation
time [8]. Although the impedance values tend to decrease with time, the
behavior is different for each strain; at high frequencies (f > 1000 Hz), the
electrical impedance decreases with time, i.e., the conductivity of the solu-
tion increases, thus generating a displacement current through the bacterial

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wall that causes the ions of the intracellular medium to contribute to the
conduction of the current [30]. Despite the plot showing a decrease in the
impedance magnitude in the first incubation hour for both bacteria, there
is not observed a direct correlation between impedance magnitude with the

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increase of bacteria population in the following hours; it may be due to the
high conductivity of solution II (0.7 S/m, Fig 3) is not significantly affected
by introducing the bacteria culture.

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For the above reasons, we focus on frequencies f < 1000 Hz; as the
frequency decreases in the 1000 Hz to 10 Hz range, the impedance values
increase until reaching a maximum value at 10 Hz. At this frequency, the
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impedance values for the two strains at t = 0 h of incubation are approx-
imately 180 Ω, while in the same electrolyte without bacteria, it is 140 Ω;
the difference in impedance values, which is 40 Ω, is related to the capaci-
tive effects associated with the bacterial walls that isolate the intracellular
medium and lead to higher solution resistance, which is reflected in the de-
crease in impedance for low frequencies [30]. Another possibility to explain
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these results is attributed to acidic groups present in the bacteria walls that
could compensate for the contractions present in the suspension, decreasing
the conductive response [31].
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3.3. Effect of Lugol in electrical properties

The addition of Lugol exhibits interesting electrical effects, as described
in this and the next sections. The impedance magnitude as a function of fre-
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quency for Staphylococcus aureus (Fig 5(a)) and Escherichia coli (Fig 5(b)),
in the presence of methyl violet and Lugol, obtained at different incubation
times, showed different behaviors; the Staphylococcus aureus strain showed
a marked progressive decrease in the magnitude of the electrical impedance
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with increasing bacterial population (Staphylococcus aureus, Fig 5(a), while

in the Escherichia coli strain, Fig 5(b), the impedance values decreased dur-
ing the first hour and then increased progressively. The observed differences
are due to the particular interaction between methyl violet, Lugol, and the
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bacterial wall of each strain [32, 33]. However, there is not observed a clear
correlation between the impedance magnitude with the incubation time. For

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this reason, it was proposed another way to analyze the data.

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(a)
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Figure 5: Bode plots of the impedance magnitudes of (a) Staphylococcus aureus (gram-
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positive) and (b) Escherichia coli (gram-negative) in PBS, TSB, methyl violet and Lugol
suspension at different incubation times.

3.4. Time evolution of the impedance magnitude

The differences in the time evolution of electrical impedance between
gram-positive and gram-negative strain observed in Fig 5 can be appreciated
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better in Fig 6 the rate of change of impedance magnitude in the incubation

time as a function of frequency.
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Figure 6: Ratio of impedance magnitude in time at different frequencies for (I) Escherichia
coli (gram-negative) and (II) Staphylococcus aureus (gram-positive) strains in the presence
of methyl violet and Lugol.
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In the frequency interval from 100 Hz to 10 Hz, a marked difference
in the behavior of the electrical impedance magnitude change ratio in each
strain is observed (Fig 6); therefore, the values of the electrical impedance
magnitude change ratio of Escherichia coli (gram-negative) are positive,
while in Staphylococcus aureus (gram-positive), the values of the electrical
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impedance magnitude change ratio are negative; in addition, when the fre-
quency decreases, it is observed that the change ratio increases in each strain.
Therefore, the results suggest that the methodology proposed here allows dif-
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ferentiation between Gram-positive and Gram-negative bacteria, by means

of the qualitative analysis of the rate of change of impedance magnitude (in
the incubation time) as a function of frequency; it is noteworthy that the
difference in the behavior mentioned above is only observed when the two
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main components of the Gram stain are present in the bacterial suspension,
i.e., methyl violet and Lugol. Another method to analyze data based on the
linear correlation coefficient is reported in Appendix D.
The differences observed in Fig 6 between Gram-positive and Gram-
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negative impedance ratios may be related to the interaction of the main

components of the Gram stain, methyl violet, and Lugol’s stain with the bac-
terial wall. Considering that the wall of Escherichia coli is less permeable
than that of Staphylococcus aureus, due to the presence of an outer mem-
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brane (Fig 1(a)) [34], a remnant concentration of methyl violet and Lugol
would remain in solution as components of the extracellular medium; thus,

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Lugol could react with charged carbon species, such as charged amino acids
from the TSB culture medium, to form uncharged oligomers. In addition, it
is possible that Lugol interacts with lipopolysaccharides [35] that are found
in large quantities as components of the cell wall of Escherichia coli (Fig
1(b)), forming more complex structures that can cause an increase in the

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magnitude of the electrical impedance of the solution; however, it is beyond
the scope of this work to provide a further explanation for the phenomenon
that causes the progressive increase in the magnitude of the impedance of
Escherichia coli with incubation time.

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4. Conclusion

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This research work contributes to the knowledge of the time evolution of
electrical properties of gram-positive and gram-negative bacteria in aqueous
suspensions with methyl violet and Lugol and establishes a method that could
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be used to differentiate Staphylococcus aureus and Escherichia coli bacteria.
The time evolution of electrical impedance spectroscopy was analyzed by
the ratio of impedance magnitude in time dZ/dt, obtaining a qualitative
different behavior for Staphylococcus aureus (gram-positive, dZ/dt < 0) and
Escherichia coli (gram-negative, dZ/dt > 0) suspended in a solution of PBS,
TSB, methyl violet and Lugol; the electrical response difference obtained
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for the two types of bacteria could be attributed to the interaction to the
characteristics between the main components of the Gram stain and the cell
wall structure. Moreover, a near-linear behavior was observed for frequencies
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lower than 200 Hz and could be used to differentiate Staphylococcus aureus

from Escherichia coli due to the ratio of impedance magnitude in time with
different signs. In spite of the inherent electrode polarization that causes
high impedance responses due to the presence of many charged components
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(metabolites, Gram reagent, inherent charge of wall bacteria), the qualitative

response obtained for each type of bacteria when is in presence of Gram
reagents is not affected.
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Author contributions
Conceptualization: Erika Viviana Godoy Alarcon, David A. Miranda.
Data curation: Erika Viviana Godoy Alarcon. Formal analysis: Erika Vi-
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viana Godoy Alarcon, Ely Dannier Valbuena Niño, David A. Miranda. In-
vestigation: Erika Viviana Godoy Alarcon, David A. Miranda. Methodology:

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Erika Viviana Godoy Alarcon, David A. Miranda. Project administration:
David A. Miranda. Supervision: David A. Miranda. Writing – original draft:
Erika Viviana Godoy Alarcon. Writing – review & editing: Ely Dannier Val-
buena Niño.

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Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the work
reported in this paper.

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Data availability
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Data will be made available on request.
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Acknowledgments
Authors appreciate the laboratory time given by the Grupo de Investi-
gación en Bioquímica y Microbiología (GIBIM) and the Laboratorio de Elec-
troquímica from the Centro de Investigación Científica y Tecnológica en Ma-
teriales y Nanociencias (CMN). E D V-Niño expresses their gratitude to the
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Colombian agency Colciencias, now MinCiencias, for the support through

doctoral scholarship 617, and to Universidad Industrial de Santander for the
support through postdoctoral position associated to project 2539.
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Appendix A. Electrode surface cleaning

Electrode cleaning was performed as described in the previous section.
Through electrochemical cleaning, the elimination of impurities on the sur-
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face of the electrodes reduces the appearance of thermoelectric effects that

alter the electrical properties of the material [27]; the last voltammetric cy-
cles observed in Fig A.7 suggest that the electrode gold surfaces were cleaned
appropriately due to the gold characteristic oxidation peaks being observed
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[21, 22, 23, 24, 25].

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Figure A.7: Analysis of the electrode surface cleaning by cyclic voltammetry: (I) First

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and (II) last voltammetric cycle (V = 100 mV /s) characteristics of a gold (Au) electrode
in a solution of sulfuric acid at a concentration of 0.5 molar (0.5 M H2 SO4 ).
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Appendix B. Current amplitude selection
Fig B.8 shows the behavior of the imaginary part of the electrical impedance
as a function of its real part (Nyquist plot); each curve corresponds to the
electrical properties measured at different amplitudes of excitation current in
PBS at a concentration of 1X. The 10.0 µA current amplitude of the excita-
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tion signal was selected because it has a low frequency with lower noise effects;
note, in Fig B.8, at current amplitude values of 0.1 µA, and 1.0 µA deviation
from the semicircle is observed, suggesting the presence of electronic noise.
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Furthermore, in electrical measurements, lower excitation is recommended

because higher current amplitudes, e.g., 40.0 µA, could produce undesired
transformations in the system [28].
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Figure B.8: Nyquist plots of PBS were obtained with different current values.
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Appendix C. Selection of the methyl violet concentration
The electrical impedance of solutions with different concentrations of
methyl violet was measured starting from 24 mM , i.e. from the concen-
tration used in the Gram staining. As the impedance magnitude decreases
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as the methyl violet concentration decreases (see Fig C.9), lower concentra-
tions tend to the electrical properties observed in the PBS electrolyte solu-
tions at a concentration of 1X and TSB culture, which are approximately
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100 Ω. For our experiments, we selected a concentration of methyl violet of

4.0 × 10−5 mM , i.e., 6 × 106 times lower than the concentration used in the
Gram staining. The reason for this choice was that the electrical impedance
magnitude values of methyl violet at different concentrations (Fig C.9) change
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the electrical properties of the solution; however, this low concentration gives
an impedance amplitude on the same order of magnitude as PBS + TSB.
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Figure C.9: Bode diagrams of the electrical impedance magnitude for a series of methyl
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violet dilutions, starting from (I) 24 mM standard solution, and dilutions (II) 1/100, (III)
1/104 , and (VI) 1/(6 × 105 ).
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Appendix D. Cell cultures growth curves

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Figure D.10: Growth curves of (I) methicillin-resistant Staphylococcus aureus (gram-
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positive) and (II) Escherichia coli O157:H7 (gram-negative) strains obtained by the plate
count method, with characteristic curves.

Appendix E. Linearity analysis

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The linear correlation coefficient (LCC) is a good parameter for analyzing

linearity; an LCC near one corresponds to linear behavior, and an LCC near
zero corresponds to nonlinear behavior. The LCCs of the magnitude of elec-
trical impedance with incubation time for excitation frequencies, Fig. E.11,
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are nearer to one for the Staphylococcus aureus strain than for Escherichia
coli. At low frequencies (f < 100 Hz), both strains have LCCs near one,
but at high frequencies (f > 1000 Hz), Escherichia coli exhibits nonlinear
behavior. The low values of LCC seen in the Escherichia coli strain at high
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frequencies (f > 1000 Hz) compared to the high values seen in the Staphylo-
coccus aureus strain may be attributed to the higher complexity phenomena
occurring between the bacterial wall, the methyl violet, Lugol and the differ-
ent components of the extracellular medium.
ep

According to these results, it can be hypothesized that the ratio of impedance

magnitude at time dZ/dt has a near-linear behavior for frequencies lower than
200 Hz.
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 ed
iew
r ev
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Figure E.11: Linear correlation coefficient as a function of frequency of the impedance
magnitude change ratio of (I) Staphylococcus aureus (gram-positive) and (II) Escherichia
coli (gram-negative) strains in the presence of methyl violet and Lugol.
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