Zeta potential as a measure of the surface

charge of mycobacterial cells

Carlos Ayala-Torres, Nicols Hernndez,

Alejandra Galeano, Lorena NovoaAponte & Carlos-Y.Soto
Annals of Microbiology
ISSN 1590-4261
Ann Microbiol
DOI 10.1007/s13213-013-0758-y

1 23

Your article is protected by copyright and

all rights are held exclusively by SpringerVerlag Berlin Heidelberg and the University
of Milan. This e-offprint is for personal
use only and shall not be self-archived in
electronic repositories. If you wish to selfarchive your article, please use the accepted
manuscript version for posting on your own
website. You may further deposit the accepted
manuscript version in any repository,
provided it is only made publicly available 12
months after official publication or later and
provided acknowledgement is given to the
original source of publication and a link is
inserted to the published article on Springer's
website. The link must be accompanied by
the following text: "The final publication is
available at link.springer.com.

1 23

Author's personal copy

Ann Microbiol
DOI 10.1007/s13213-013-0758-y

ORIGINAL ARTICLE

Zeta potential as a measure of the surface charge

of mycobacterial cells
Carlos Ayala-Torres & Nicols Hernndez & Alejandra Galeano &
Lorena Novoa-Aponte & Carlos-Y. Soto

Received: 28 August 2013 / Accepted: 29 October 2013

# Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract The surface charge of bacteria is closely related to

their envelope structure and interactions with surfaces in natural environments. The aim of this study was to estimate the
effect of experimental conditions on the zeta () potential of
mycobacterial cells as a measure of their cell-surface charge.
We observed that Mycobacterium smegmatis mc2155 cells at
physiological conditions displayed a high and stable potential (42.9 5.9 mV) which increased from the lateexponential phase of growth and at pH levels of >8.0. The
optimal conditions for estimating the surface charge of
mycobacteria using the potential occurred when cells were
harvested during the exponential growth phase (OD595 0.3
0.5) and then dispersed in solutions with pH levels of 7.0
10.0. These optimal conditions of potential measurements
were useful for differentiating between the virulent M. tuberculosis H37Rv strain and various non-virulent mycobacterial
strains at pH 9.8. This study is the first to use zetametry to
estimate the cell-surface charge of M. tuberculosis cells. We
expect that the experimental conditions presented in this work
will have further applications to estimate the cell-surface
charge of other wild-type or genetically modified mycobacterial species and thereby further our understanding of the
physicochemical interactions of mycobacteria with external
surfaces in natural environments.

Keywords Mycobacterium smegmatis mc2155 .

Mycobacterium tuberculosis . Zeta potential . Electrophoretic
mobility . Cell-surface charge
C. Ayala-Torres : N. Hernndez : A. Galeano : L. Novoa-Aponte :
C.<Y. Soto (*)
Chemistry Department, Faculty of Sciences, Universidad Nacional
de Colombia, Carrera 30 N 45-03, Ciudad Universitaria, Bogot,
Colombia
e-mail: cysotoo@unal.edu.co

Introduction
The surface charge is a physicochemical property that is
associated with the composition of the cell envelope, and it
plays an important role in the interaction of bacteria with ions,
particles and surfaces. The cell-surface charge affects the entry
of metabolites into the cells and the bacterias interaction with
cellular receptors exposed by hosting cells (Yeung and
Grinstein 2007; Goulter et al. 2009; Bar-Even et al. 2011).
Bacteria normally display a negative surface charge under
physiological conditions (van Loosdrecht et al. 1987). The
surrounding environment can, however, influence the cellsurface charge of bacteria, and a substantial increase in the
ionic strength of the environment decreases the interaction of
the bacteria with external surfaces (Mozes et al. 1988). Consequently, bacterial adhesion to external surfaces has been
understood in terms of electrostatic interactions (van
Loosdrecht et al. 1987; Holder et al. 2007; Seale et al. 2010).
The genus Mycobacterium comprises both pathogenic species, such as M. tuberculosis and M. leprae, which are the
etiologic agents of tuberculosis and leprosy, respectively, and
saprophytic species, such as M. smegmatis, which is an environmental mycobacteria common in water and soil. In addition, the genus Mycobacterium is characterized by a high lipid
content in the cell envelope that reduces mycobacterial permeability and the substances exchange with the environment
(Daffe and Draper 1998). The short generation time of M.
smegmatis facilitates this species as a model for the study of
physiological and morphological characteristics of
mycobacteria (Altaf et al. 2010; Birch et al. 2010).
Determination of the cell-surface charge requires laborious
experimentation that involves the evaluation of many methodological variants. However, this physicochemical parameter
can be deduced from the electrokinetic or zeta () potential,
which can be estimated from the electrophoretic mobility of
cells (Hiemenz and Rajagopalan 1997). In zetametry

Author's personal copy

Ann Microbiol

experiments, a cellular dispersion with a determined ionic

strength is placed into an electrophoresis glass capillary tube
that is subjected to a potential difference. The time that cells
spend migrating under a constant electric field is related to
their electrophoretic mobility (van der Mei and Busscher
2001; Rosenhahn et al. 2009). The potential and electrophoretic mobility of cells are influenced by the cell surface charge,
electric field, temperature and ionic conditions of the cellular
dispersion (van Loosdrecht et al. 1987). For example, it has
been observed that the potential of Escherichia coli cells is
influenced by the ionic strength and pH of the bacterial
dispersion and especially by the presence or absence of a
bacterial capsule (Bayer and Sloyer 1990).
Previous studies using zetametry have shown that the surface charge of the Mycobacterium bovis BCG, Tice substrain
is negatively charged (Zhang et al. 1988; Kristensen et al.
1992). There is as yet no optimized protocol for determining
the potential of mycobacterial cells and, additionally, the cell
surface charge of M. tuberculosis cells is still unknown. We
have assessed the optimal conditions to determine the potential of mycobacterial cells as a function of cell viability, the
number of cells, their growth phase and the pH of the dispersing solution, using M. smegmatis mc2155 as a working strain.
The optimal conditions observed for M. smegmatis cells were
useful for differentiating the virulent M. tuberculosis H37Rv
strain from various non-virulent mycobacterial strains.

Material and methods

Mycobacterial strains and growth conditions
The saprophyte strain M. smegmatis mc2155 (ATCC 700084)
(Snapper et al. 1990), the vaccine strain M. bovis BCG Pasteur
1137P2 and the M. tuberculosis reference strains H37Rv
(ATCC 27294) and H37Ra (ATCC 25177) were used in this
study. Mycobacterial cells were cultured at 37 C either in
Middlebrook 7H9 (subsequently referred to as 7H9) broth
(in mol L1: NaCl, 0.14; KCl, 0.005; Na2HPO4, 0.01;
KH2PO4, 0.002) supplemented with ADC [0.5 % (w/v)
bovine serum albumin, 0.2 % (w/v) dextrose, 0.085 % (w/v)
NaCl, 0.0003 % (w/v) beef catalase and 0.05 % (v/v) glycerol]
or on 7H10-OADC plates [ADC + 0.05 % (v/v) oleic acid]. To
avoid cellular agglomeration, the mycobacterial culture media
were supplemented with 0.05 % Tween 80. To obtain dead
cells, mycobacteria that were grown on 7H10-OADC plates
were exposed to UV irradiation at 302 nm and 40 W for
560 min using a DyNA Light transilluminator (Labnet,
Edison, NJ). The cells were scraped, and the viability of
irradiated mycobacteria was monitored by incubating an
aliquot of cells in 7H9 broth at 37 C with vigorous agitation
for 3 days in the case of fast-growth mycobacteria or for 4 weeks
for slow-growth mycobacteria. To estimate the amount of dead

mycobacteria in the samples, the OD595 of cellular dispersions

was interpolated in curves correlating the colony-forming units
(CFU) and the OD595 of cultures. The CFU of mycobacteria
was calculated by plating serial dilutions of cultures on 7H10OADC plates and incubating them at 37 C.
Dispersing solution
The zetametry experiments were performed using bacteria
suspended in 1X phosphate buffered saline (PBS; in
mol L1: NaCl, 0.14; KCl, 0.005; Na2HPO4, 0.01; KH2PO4,
0.002; pH 7.0), which has a calculated ionic strength of
0.177 mol L1. This dispersing solution was kept constant
for the optimization experiments. To estimate the influence of
pH, we performed individual experiments using dispersing
solutions that were adjusted from pH 3.0 to 11.0.
Zeta potential and electrophoretic mobility
Measurement of the potential and electrophoretic mobility
of cells was performed simultaneously on a Zetameter 3.0+
instrument (Zeta-Meter Inc., Staunton, VA) with a cell constant of 66 cm1. This apparatus consists of a glass capillary
used as an electrophoretic chamber, and the time it took for
cells to migrate to the anode was related to their electrophoretic mobility measurement. For initial optimization, the
zetametry experiments were performed using M. smegmatis
mc2155 cells harvested at the exponential growth phase. To
establish the optimal conditions for zetametry measurements,
we first washed and then resuspended an initial amount of
approximately 1.2108 CFU mL1 live or dead mycobacteria
in 40 mL of dispersing solution; this cellular dispersion was
then diluted 1:4 in deionized water and vigorously vortexed to
disrupt cellular clumps. The cell dispersions were then placed
into the glass capillary, and the time cells spent migrating to
the anode was monitored by optical microscopy on the
zetameter. The zetametry measurements were performed at
150 V and 20 C, and three independent experiments were
performed in which refractive and bacterial absorption indexes of 1.58 and 0, respectively, were considered. Each reported
potential (mV) or conductance value (S cm1), as calculated
by the Zetameter 3.0+ instrument, was the average of 20
technical replicas with a standard deviation (SD) of <5 %.
This basic protocol was then used with live cells of M.
smegmatis mc2155 to evaluate how the number of cells, the
growth phase and the dispersing solution pH affected potential values.
Variations in the cellular phase of growth
The influence of the growth phase on potential values was
evaluated for M. smegmatis cells harvested from the latent to
the stationary growth phases (from OD595 =0.054 to OD595 =

Author's personal copy

Ann Microbiol

1.026). Samples were collected every 24 h, washed with 1X

PBS and resuspended in the same buffer, and then the basic
protocol previously described for potential measurements of
M. smegmatis cells was applied.
Variations of pH
Measurement of potential as a function of pH was assessed
using the basic protocol for M. smegmatis cells but varying
the pH of the dispersing solution from pH 3.0 to 11.0 using
0.1 mol L1 HCl or 0.1 mol L1 NaOH to achieve similar ionic
strength in the samples.

Fig. 1 Mycobacterium smegmatis mc2155 survival and growth in culture. Optical density at 595 nm (OD595) vs. colony-forming units (CFU)
mL1 for M. smegmatis grown in standard conditions of culture. Bars
Standard deviation (SD) from three independent experiments performed
in duplicate

Variations in the cell concentration

Measurement of the potential was also performed while
varying the cell concentration of M. smegmatis from 4.5
107 to 1.8108 CFU mL1 in 40 mL of the dispersing solution
and applying the basic protocol described above.
Neutral red uptake test
The capacity of mycobacterial cells to fix the basic dye neutral
red was assessed as reported previously (Soto et al. 2002).
Briefly, mycobacterial cells were placed in screw-cap tubes
containing 5 mL of 50 % aqueous methanol and then incubated for 1 h at 37 C. The fluid was removed, and the cells
were washed as described above. The washed cells were then
resuspended in 5 mL of 0.002 % neutral red in TrisHCl
buffer, pH 9.8, and incubated at room temperature for 24 h.

Results and discussion

Establishing a basic protocol for potential measurements
of mycobacterial cells
Based on the results of zetametry experiments previously
reported for other bacterial systems (Ciesla et al. 2011), we
performed the measurement of potential at room temperature (approx. 20 C). This working temperature ensured
shorter times for sample preparation, which increased the
reproducibility of the results. The initial amount of
mycobacteria used, approx. 1.2108 CFU mL1, was obtained from 1 mL of cells cultured in 7H9 broth that were
harvested at an OD595 =0.3 (Fig. 1). Cellular clumping was
prevented by supplementing the cultures with 0.05 % Tween
80 and by vigorous vortexing of cellular dispersions. The
presence of possible clumps was monitored by an optical
microscopy system that was coupled with the Zetameter +
3.0 instrument during the zetametry measurements.
In general, the measurements of potential were reproducible (SD < 5 %) as early as 24 h after the cellular dispersions of

mycobacterial cells were prepared. Serial dilutions of M.

smegmatis dispersions (1.2108 cells in 40 mL of dispersing
solution) to a concentration of 1:100 (v/v) did not produce
significant changes in the potential that affected the reproducibility of measurements (data not shown). The samples
prepared following this protocol did not show significant
differences in the specific conductance, which ensured a similar ionic environment for the zetametry measurements.
The surface charge of M. smegmatis depends on the metabolic
stage of cells
Based on growth kinetics observed for M. smegmatis mc2155
cells (Fig. 1), the potential was measured on cells harvested
from the latent to stationary phases of growth. A significant
increase of negative charges on the cell surface of M.
smegmatis cells at physiological pH (7.0) was observed when
cells entered the exponential phase of growth (Figs. 1, 2);
however, the highest values were observed for cells harvested
at the stationary phase of growth. The reproducibility of
results (p =95 %) was constant in all growth phases (Fig. 2).
Mycobacteria at the stationary phase, where cellular death
is higher, have an increased leakage of charged molecules into
the environment (Hayashi et al. 2003). This phenomenon
produces an increased ionic concentration in the layer immediately external to the cell surface, thus increasing the electrical conductivity and potential. For methodological purposes, it is recommended that measurements of potential
and electrophoretic mobility for M. smegmatis cells be performed at the late-exponential phase of growth, specifically at
an OD595 of 0.30.6 (Figs. 1, 2). At this growth phase,
adequate bacterial fitness is guaranteed, and high values of
potential and electrophoretic mobility indicate cellular dispersion stability.
Effect of pH on the potential of M. smegmatis cells
The potential of M. smegmatis cells (at the exponential
phase), as measured at different pH levels, is shown in

Author's personal copy

Ann Microbiol

Fig. 2 Relation between the growth phase and the electrokinetic or zeta
() potential of M. smegmatis mc2155 cells

Fig. 3. Briefly, the cell-surface charge of cells was relatively

constant from pH 6.0 to 8.0 (approximately 28 mV), and
increased at pH >8.0. The highest values of potential and
electrophoretic mobility were registered at pH=10.0 [44.4
2.62 mV and 3.430.35 (microns s1) (V cm1), respectively], indicating high stability of the cellular dispersion in
this pH range. By contrast, low values were observed when
measurements were performed in the range pH 3.05.0
(Fig. 3). We also observed that mycobacterial dispersions at
pH levels of <3.0 and >10.0 displayed an increased viscosity
due to cellular lysis.
The potential and electrophoretic mobility markedly
decreased at acidic pH levels as a consequence of negative
cell-surface charge neutralization by the excess of protons
(H+) in the surrounding environment. In natural conditions,
M. smegmatis cells are sometimes found in acidic water and
soils (Livanainen 1995), and M. tuberculosis cells face an acid
pH environment inside macrophages (Sturgill-Koszycki et al.
1994). Thus, an acidic pH in natural environments promotes
the neutralization of the negative cell-surface charge of
mycobacteria. Taking account of the changes of mycobacterial
viability as a function of pH (Chapman and Bernard 1962), we
recommend that zetametry experiments for mycobacterial
cells be performed at a pH between 7.0 and 10.0, to obtain
constant and relatively high values of potential.
Effect of cell concentration on the potential of M. smegmatis
cells

values of potential and electrophoretic mobility increased

in parallel to increasing cell concentration. Although the ionic
strength and pH remained constant in the samples, cells in
concentrated mycobacterial dispersions displayed a higher net
cell-surface charge as compared to cells in less concentrated
dispersions. In dilute cellular dispersions (CFU mL1 <9.0
107), the cell migration was less reproducible and stable, as
corroborated by the standard deviations of the migrations
(Fig. 4). However, some cell aggregation was observed in
concentrated cellular dispersions (CFU mL1 >1.5108), generating cell deposition on electrodes that altered the potential
values. For technical reasons, the values of potential and
electrophoretic mobility are more reproducible using mycobacterial dispersions of between 9.0 10 7 and 1.5
108 CFU mL1.

The surface charge of M. smegmatis cells as a function of cell

viability
Mycobacterial cells were completely dead after irradiation
with UV light for more than 60 min. Using the basic protocol,
live and dead cells of M. smegmatis mc2155 showed a
potential of 42.52.9 and 49.63.9 mV, respectively. These high values of the potential indicate a great stability
between the cell-surface charge and the ionic strength of the
dispersing solution in both cellular conditions. However, dead
cells of M. smegmatis displayed an increased amount of
negative charges on the cell surface compared to the live cells.
Unlike other previous studies (Soni et al. 2008; Ciesla et al.
2011), the method for killing cells used in this work did not
introduce extensive changes in cell envelope integrity that
could alter the structure of the surface molecules. Consequently, the difference observed in the potential between the live
and dead M. smegmatis cells could be due to changes in the
metabolic activity of the mycobacteria. Live mycobacteria
have functional transport systems (efflux pumps, enzymes,
ion exchange, proton pumps, etc.) across the cell envelope
that contribute to an exchange of electrolytes and metabolites
which preserves the ion homeostasis (Agranoff and Krishna
2004; Amaral et al. 2007; Rao et al. 2008). In contrast, ion

The dependence of the cell-surface charge of M. smegmatis

on mycobacterial concentration (Fig. 4) showed that the

Fig. 3 Effect of pH on the potential of M. smegmatis mc2155 cells

Fig. 4 Effect of cell concentration on the potential of M. smegmatis

mc2155 cells

Author's personal copy

Ann Microbiol
Table 1 Zeta potential and electrophoretic mobility of mycobacterial cells at physiological conditionsa
Zeta potential and electrophoretic mobility Mycobacterium smegmatis mc2155 M. tuberculosis H37Rv M. tuberculosis H37Ra M. bovis BCG
Zeta potential (mV)
Electrophoretic mobility
(microns s1) (V cm1)
Specific conductivity (S cm1)

29.7
2.58

22.6
2.09

24.3
2.13

28.1
2.38

1547

1535

1238

1427

The reported values were calculated using a Zetameter 3.0+ instrument and are the average of 20 separate measurements for each sample, with a standard
deviation (SD) of <5 %
Mycobacteria harvested at the exponential phase of growth (OD595 =0.30) were dispersed in 1X phosphate buffered saline (PBS) (1.2108 CFU mL1 )
at pH 7.0. In all cases, the glass cell factor (K) was 66 cm1 . The specific conductance obtained corroborates a similar ionic environment in all samples

transport in dead cells is inactive, causing a leakage of charged

molecules that remain on the cell surface (Eboigbodin et al.
2006). This phenomenon may partially explain the augmented
potential values for dead cells that were observed in our
study.
Cell-surface charge of M. tuberculosis cells
In this study, the optimal conditions that were found using M.
smegmatis cells were then used to measure the potential and
electrophoretic mobility of M. tuberculosis cells, and these
results were compared with those obtained for other nontuberculous mycobacterial cells used in our study. Our review
of the literature revealed that our study is the first in which
zetametry experiments have been performed on M. tuberculosis cells. For biosecurity management precautions, the nonviability of the pathogenic H37Rv cells was corroborated prior
to performing the zetametry measurements; similar to our
observations for M. smegmatis, the cells of M. tuberculosis
and M. bovis BCG that were irradiated with UV light for
60 min did not grow in 7H9-ADC broth when incubated at
37 C with agitation.
Cells of either the virulent H37Rv or the attenuated H37Ra
M. tuberculosis strains which were harvested at the exponential phase of growth (OD595 =0.3) and then dispersed in 1X
PBS (1.2108 CFU mL1) at pH 7.0 did not show significant
differences in the cell-surface charge at pH7.0 (Table 1).
However, the absolute value of the potential and the

electrophoretic mobility for the M. tuberculosis cells

(H37Rv and H37Ra) were approximately 19 and 15 % lower
than those of the non-tuberculous strains M. bovis BCG and
M. smegmatis mc2155, respectively (Table 1). This result
indicates that the M. tuberculosis strains contain a higher
proportion of positive charges on the cell surface at physiological pH as compared to non-tuberculous strains (Table 1).
The cell-surface charge associated with the cell-wall composition is known to determine the ability of M. tuberculosis
cells to react with basic dyes (Middlebrook et al. 1959).
Therefore, virulent M. tuberculosis strains have the capacity
to protonate the neutral red stain in an alkaline aqueous
environment (pH=9.8), resulting in the cells being stained
red (Dubos and Middlebrook 1948; Soto et al. 2002;
Cardona et al. 2006). We performed zetametry experiments
at pH=9.8 on cells of M. tuberculosis H37Rv strain, using the
M. tuberculosis H37Ra strain and the non-tuberculous M.
bovis BCG and M. smegmatis mc2155 strains as controls.
As shown in Table 2, at pH 9.8, cells of M. tuberculosis
H37Rv displayed approximately 29 % fewer negative charges
on the cell surface compared with the control strains, with the
result that the alkaline pH increased the proportion of positive
charges on the cell surface of the virulent H37Rv strain. We
postulate that this higher proportion of positive charges on the
cell surface of strain H37Rv could be due in part to the
presence of H+ ions associated with certain fatty acids and
glycolipids in the cell envelope of virulent M. tuberculosis
strains that are known virulence factors (Daffe and Etienne

Table 2 Zeta potential and electrophoretic mobility of mycobacterial cells at pH 9.8a

Zeta potential and electrophoretic mobility
Zeta potential (mV)
1

Electrophoretic mobility (microns s ) (V cm )

Specific conductivity (S cm1)
Neutral red stainingb

M. tuberculosis H37Rv

M. tuberculosis H37Ra

M. bovis BCG

M. smegmatis mc2155

25.9

33.0

30.9

45.7

2.40
1740
+

2.59
1208

2.53
1464

3.34
1523

Mycobacteria harvested at the exponential phase of growth (OD595 =0.35) were dispersed in 1X PBS (1.2108 CFU mL1 ) at pH 9.8. In all cases, the
glass cell factor (K) was 66 cm1 . The specific conductance obtained corroborates a similar ionic environment in all samples
a

The reported values were calculated using a Zetameter 3.0+ instrument and are the average of 20 separate measurements for each sample, with a SD of <5 %

+, Red staining of cells by neutral red protonation; , no staining of the cells by their inability to protonate neutral red

Author's personal copy

Ann Microbiol

1999; Forrellad et al. 2013). This higher proportion of H+ ions

increases the acidity at the cell surface of the virulent strain,
thus facilitating protonation of the neutral red dye at an
alkaline pH (Table 2) (Soto et al. 2002).
The potential values obtained for M. tuberculosis H37Ra
and M. bovis BCG suggest that non-virulent mycobacteria do
not retain sufficient H+ on their cell surface at an alkaline pH
to protonate the neutral red stain (Table 2). The same outcome
was obtained for the saprophyte M. smegmatis mc2155, which
displayed a lesser proportion of positive charges on the cell
surface and, therefore, in contrast to M. tuberculosis H37Rv,
had the lowest capacity to protonate neutral red. Thus, the cellsurface charge estimated by the potential was useful for
differentiating the virulent M. tuberculosis H37Rv strain from
various non-virulent mycobacterial strains at pH 9.8.

Conclusions
The experimental results obtained in this work indicate that
zetametry is a useful method for estimating the cell-surface
charge of mycobacterial cells. For methodological purposes,
we recommend that measures of potential and electrophoretic mobility be performed using mycobacterial cells harvested at an OD595 ranging between 0.3 and 0.5 and dispersed in
solutions with a pH level of 7.010.0. Mycobacterial cells
cultured under standard conditions displayed a high and stable
negative cell-surface charge, which increased from the stationary phase of growth in cultures at a pH range of 8.010.0
and in cell dispersions at a concentration of >1.1
108 CFU mL1. In addition, this work demonstrates that
potential measurements are useful for differentiating the virulent M. tuberculosis H37Rv strain from other non-virulent
mycobacteria at pH 9.8.
Acknowledgments This work was supported by the Divisin de
Investigacin Bogot (DIB)Universidad Nacional de Colombia, grants
14337, 15084 and 16060. The authors thank the Laboratory of Chemical
Engineering at the Universidad Nacional de Colombia for its valuable
help in the zetametry experiments.

References
Agranoff D, Krishna S (2004) Metal ion transport and regulation in
Mycobacterium tuberculosis. Front Biosci 9:29963006
Altaf M, Miller CH, Bellows DS, OToole R (2010) Evaluation of the
Mycobacterium smegmatis and BCG models for the discovery of
Mycobacterium tuberculosis inhibitors. Tuberculosis (Edinb) 90(6):
333337. doi:10.1016/j.tube.2010.09.002
Amaral L, Martins M, Viveiros M (2007) Enhanced killing of intracellular multidrug-resistant Mycobacterium tuberculosis by compounds
that affect the activity of efflux pumps. J Antimicrob Chemother
59(6):12371246. doi:10.1093/jac/dkl500
Bar-Even A, Noor E, Flamholz A, Buescher JM, Milo R (2011)

Hydrophobicity and charge shape cellular metabolite concentrations. PLoS Comput Biol 7(10):e1002166. doi:10.1371/journal.
pcbi.1002166
Bayer ME, Sloyer JL Jr (1990) The electrophoretic mobility of gramnegative and gram-positive bacteria: an electrokinetic analysis. J
Gen Microbiol 136(5):867874
Birch HL, Alderwick LJ, Appelmelk BJ, Maaskant J, Bhatt A, Singh A,
Nigou J, Eggeling L, Geurtsen J, Besra GS (2010) A truncated
lipoglycan from mycobacteria with altered immunological properties. Proc Natl Acad Sci USA 107(6):26342639. doi:10.1073/pnas.
0915082107
Cardona PJ, Soto CY, Martin C, Giquel B, Agusti G, Andreu N, Guirado
E, Sirakova T, Kolattukudy P, Julian E, Luquin M (2006) Neutralred reaction is related to virulence and cell wall methyl-branched
lipids in Mycobacterium tuberculosis. Microbes Infect 8(1):183
190. doi:10.1016/j.micinf.2005.06.011
Chapman JS, Bernard JS (1962) The tolerances of unclassified
mycobacteria. I. Limits of pH tolerance. Am Rev Respir Dis 86:
582583
Ciesla J, Bieganowski A, Janczarek M, Urbanik-Sypniewska T (2011)
Determination of the electrokinetic potential of Rhizobium
leguminosarum bv trifolii Rt24.2 using Laser Doppler
Velocimetrya methodological study. J Microbiol Methods 85(3):
199205. doi:10.1016/j.mimet.2011.03.004
Daffe M, Draper P (1998) The envelope layers of mycobacteria with
reference to their pathogenicity. Adv Microb Physiol 39:131203
Daffe M, Etienne G (1999) The capsule of Mycobacterium tuberculosis
and its implications for pathogenicity. Tuber Lung Dis 79(3):153
169. doi:10.1054/tuld.1998.0200
Dubos RJ, Middlebrook G (1948) Cytochemical reaction of virulent
tubercle bacilli. Am Rev Tuberc 58(6):698
Eboigbodin KE, Newton JR, Routh AF, Biggs CA (2006) Bacterial
quorum sensing and cell surface electrokinetic properties. Appl
Microbiol Biotechnol 73(3):669675. doi:10.1007/s00253-0060505-4
Forrellad MA, Klepp LI, Gioffre A, Sabio y Garcia J, Morbidoni HR, de
la Paz Santangelo M, Cataldi AA, Bigi F (2013) Virulence factors of
the Mycobacterium tuberculosis complex. Virulence 4(1):366. doi:
10.4161/viru.22329
Goulter RM, Gentle IR, Dykes GA (2009) Issues in determining factors
influencing bacterial attachment: a review using the attachment of
Escherichia coli to abiotic surfaces as an example. Lett Appl
Microbiol 49(1):17. doi:10.1111/j.1472-765X.2009.02591.x
Hayashi H, Seiki H, Tsuneda S, Hirata A, Sasaki H (2003) Influence of
growth phase on bacterial cell electrokinetic characteristics examined by soft particle electrophoresis theory. J Colloid Interface Sci
264(2):565568. doi:10.1016/S0021-9797(03)00418-1
Hiemenz P, Rajagopalan R (1997) Principles of colloid and surface
chemistry. Third edition, revised and expanded. Marcel Dekker,
New York
Holder DJ, Kirkland BH, Lewis MW, Keyhani NO (2007) Surface
characteristics of the entomopathogenic fungus Beauveria
(Cordyceps) bassiana. Microbiology 153(Pt 10):34483457. doi:
10.1099/mic.0.2007/008524-0
Kristensen S, Tian Y, Klegerman ME, Groves MJ (1992) Origins of BCG
surface charge: effect of ionic strength and chemical modifications
on zeta potential of Mycobacterium bovis BCG, Tice substrain,
cells. Microbios 70(284285):185198
Livanainen E (1995) Isolation of mycobacteria from acidic forest soil
samples: comparison of culture methods. J Appl Bacteriol 78(6):
663668
Middlebrook G, Coleman CM, Schaefer WB (1959) Sulfolipid from
virulent tubercle bacilli. Proc Natl Acad Sci USA 45(12):18011804
Mozes N, Leonard AJ, Rouxhet PG (1988) On the relations between the
elemental surface composition of yeasts and bacteria and their
charge and hydrophobicity. Biochim Biophys Acta 945(2):324334

Author's personal copy

Ann Microbiol
Rao SP, Alonso S, Rand L, Dick T, Pethe K (2008) The proton motive
force is required for maintaining ATP homeostasis and viability of
hypoxic, nonreplicating Mycobacterium tuberculosis. Proc Natl
Acad Sci USA 105(33):1194511950. doi:10.1073/pnas.
0711697105
Rosenhahn A, Finlay JA, Pettit ME, Ward A, Wirges W, Gerhard R,
Callow ME, Grunze M, Callow JA (2009) Zeta potential of motile
spores of the green alga Ulva linza and the influence of electrostatic
interactions on spore settlement and adhesion strength.
Biointerphases 4(1):711. doi:10.1116/1.3110182
Seale RB, Bremer PJ, Flint SH, McQuillan AJ (2010) Characterization of
spore surfaces from a Geobacillus sp. isolate by pH dependence of
surface charge and infrared spectra. J Appl Microbiol 109(4):1339
1348. doi:10.1111/j.1365-2672.2010.04760.x
Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr (1990)
Isolation and characterization of efficient plasmid transformation
mutants of Mycobacterium smegmatis . Mol Microbiol 4(11):
19111919
Soni KA, Balasubramanian AK, Beskok A, Pillai SD (2008) Zeta potential of selected bacteria in drinking water when dead, starved, or
exposed to minimal and rich culture media. Curr Microbiol 56(1):
9397. doi:10.1007/s00284-007-9046-z

Soto CY, Andreu N, Gibert I, Luquin M (2002) Simple and rapid

differentiation of Mycobacterium tuberculosis H37Ra from M. tuberculosis clinical isolates through two cytochemical tests using
neutral red and nile blue stains. J Clin Microbiol 40(8):30213024
Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins
HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG (1994)
Lack of acidification in Mycobacterium phagosomes produced by
exclusion of the vesicular proton-ATPase. Science 263(5147):678
681
van der Mei HC, Busscher HJ (2001) Electrophoretic mobility distributions of single-strain microbial populations. Appl Environ Microbiol
67(2):491494
van Loosdrecht MC, Lyklema J, Norde W, Schraa G, Zehnder AJ (1987)
Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion. Appl Environ Microbiol
53(8):18981901
Yeung T, Grinstein S (2007) Lipid signaling and the modulation of
surface charge during phagocytosis. Immunol Rev 219:1736. doi:
10.1111/j.1600-065X.2007.00546.x
Zhang A, Groves MJ, Klegerman ME (1988) The surface charge of cells
of Mycobacterium bovis BCG vaccine, Tice substrain. Microbios
53(216217):191195