FEMS Microbiology Letters 12 (1980) 365-367
Published by Elsevier Biomedical Press
365
Electrostatic interaction chromatography, a method for
assaying the relative surface charges of bacteria
Karsten Pedersen
Department of Marine Microbiology, Botanical Institute, Universi O, of GOteborg, Carl Skottsbergs Gata 22, S-413 19 G6teborg, Sueden
Received 6 August 198 I
Accepted 18 August 1981
1. INTRODUCTION
Electrostatic and hydrophobic forces are often
pointed out as important factors involved in microbial interactions at interfaces [11. The hydrophobic interaction chromatography (HIC) described by Hjert~n et al. [21 has been used by
Dahlb~ick et al. [3] to assay the relative hydrophobicity of marine bacteria. The net surface
charge of bacteria can be determined by measuring their electrophoretic mobility [4], but that is a
rather laborious method compared to the ESIC
method described in the present paper.
Bacteria can be looked upon as macro-ions and
the adsorption of bacteria to ion exchange resins
have been studied by several authors and the
results reviewed by Daniels [5]. Ion exchange resins are either positively or negatively charged
polymeric lattices in association with small dissociable counter ions. The bacterial affinity for the
ion exchanges in electrostatic interaction chromatography (ESIC) is dependent on the surface
charges of the assayed bacteria. Investigators
primarily interested in the relative values of the
surface charges, have a time-saving advantage in
using ESIC instead of particle electrophoresis.
2. MATERIALS AND METHODS
25 isolates of marine bacteria, sampled and
assayed for their relative hydrophobicity by
Dahlb~ick et al. [3], were characterized with respect
to relative surface charges by means of ESIC. The
nine salt solution (NSS) used in ESIC consists of
NaC1 23.48g; Na2SO4 1.96g; NaHCO 3 0.10g;
KC1 0.33g; KBr 0.05g; MgC12.2H20 2.49g;
CaC12 • 2 H20 0.55 g; SrCI 2 • 6 H 2 0 0.01 g; H3BO3
0.01 g; double distilled water 1000 ml, pH 8.2.
In the electrostatic interaction chromatography
Pasteur pipettes (diam. 5 mm) were plugged with
glass wool and washed with ethanol and a 75%
NSS. 1 g of the anion exchange resin Dowex 1 × 8,
or 1 g of the cation exchange resin Dowex 50W × 8,
both of analytical grade and a mesh-size of
100/200 (80-150 #m) (Serva, Heidelberg), was
suspended in 1 ml of 75% NSS and packed in the
Pasteur pipettes. The procedure was thereafter as
described by Dahlb~ick et al. [3] for HIC, applying
1-ml portions of a 75% NSS containing about 109
metabolically 3H-labeled and washed cells to the
resin and the gel columns, and eluting with 4 X 3 ml
75% NSS.
The affinity of the bacteria for the ion exchange
resins, or for the hydrophobic Octyl Sepharose
C1-4B gel (Pharmacia, Uppsala), is expressed as
the ratio between the radioactivity of the resin
fraction and the corresponding eluate (r/e), or the
gel fraction and the corresponding eluate (g/e).
Increasing r / e values indicate an increasing
amount of surface charges of the bacteria assayed.
Increasing g / e values indicate an increasing degree of bacterial surface hydrophobicity. Repeated
0378- t097/81/0000-0000/$02.75 g~' 1981 Federation of European Microbiological Societies
366
assays showed good agreement in the r / e and g / e
values.
3. RESULTS AND DISCUSSION
When performing studies of bacterial adsorption to ion exchange resins as in ESIC, several
factors that can affect the adsorption have to be
considered: (a) The size of the resin particles must
be large enough to avoid a filtration effect. The
low r / e and g / e values of the isolates B12 and
B18 (Table 1) reveal that the filtration effect in
ESIC is negligible with the mesh-size used. (b) The
pH and ionic strength [6] have to be kept at
defined values. (c) The packing of the Pasteur
pipettes, the elution rate, and the handling of the
Table I
r / e and g / e values for 25 isolates of marine bacteria
The isolates have been grouped according to increasing affinity
for the anion exchange resin.
Isolate
Anion exchange
resin r / e values
Cation exchange
resin r / e values
Octyl
sepharose
g / e values
B 12
B 18
B 32
B 19
B 31
B 133
B 16
B 34
B 35
B 55
Y 84
B 102
B 96
Y 125
Y 57
B 123
Y 55
Y 66
B 112
B 1
B 1I 1
Y 77
B 104
B 86
Y 59
0.02
0.03
0.23
0.34
0.35
0.50
0.68
1.87
2.15
6.27
10.3
10.6
12.8
12.9
16.7
17.9
41.0
41.5
43.5
53.1
59. I
65.0
83.7
132
205
0.06
0.07
0.18
0.24
3.54
0.62
6.00
0.86
0.35
1.51
0.47
4.80
1.21
0.22
0.82
0,39
0,22
0.15
0.72
0.32
0,05
1.06
0.26
0.16
0.11
0.01
0.03
0.52
1.04
1.32
2.36
1.60
4.44
2.16
7.94
14.2
45.9
39.0
0.81
3.70
71.3
2.68
0.68
2,16
0.34
0.94
0.26
1.36
1.44
0.68
isolates have to be standardized. (d) The exchange
capacity of the resins must be sufficiently large. It
is limited by the total surface area of the resin
particles. If a monolayer of cells is adsorbed to the
surface of a 100/200 mesh resin, the theoretical
number of bacteria that can be adsorbed approaches 10 l° cells/g resin. This capacity figure
has been calculated and experimentally verified by
Daniels and Kempe [7]. In the present work 109
cells/g resin have been used, ensuring a load well
below the capacity limit.
Neihof and Loeb [8], using particle electrophoresis, have shown that particulate matter in sea
water has a net negative surface charge. This is
true also for marine bacteria as shown by the
present ESIC results were all anion r / e values,
whether high or low, corresponded to low cation
r / e values (Table 1), thus indicating that the isolated bacteria had net surface charges ranging
from about zero to strongly negative.
The higher the charge of the bacteria surface,
the less the possibility of detecting a hydrophobic
property, if present. This might explain why none
of the isolated bacteria exhibited a high net negative surface charge together with a high degree of
hydrophobicity (Table 1). Hydrophilic bacteria
possessing hydrophobic sites on the cell surface, as
those observed by Marshall and Cruickshank [9],
may, if the hydrophilic parts are negatively
charged, adsorb both to the hydrophobic gel and
to the anion exchange resin as was the case with
the isolates Y84, B102, B96, and B123 (Table 1).
The isolates B12 and B18 did not adsorb to any of
the resins nor to the gel indicating that they were
hydrophilic and uncharged.
By means of ESIC and HIC methods it is
possible to assay a combination of two of the
surface properties which enables bacteria to interact at most interfaces.
ACKNOWLEDGEMENTS
This work was supported by the Swedish National Board of Energy Source Development grants
5565 061. I would like to thank Mr. Anders
M~rtensson for excellent technical assistance and
Prof. Birgitta Norkrans for valuable discussions.
367
REFERENCES
[l] Marshall, K.C. (1976) Interfaces in Microbial Ecology,
Harvard University Press, Cambridge, MA.
[2] Hjert+n, S., Rosengren, J. and P~man, S. (1974) J. Chromatogr. 101,281-288.
[3] Dahlb~ck, B., Hermansson, M., Kjelleberg, S. and Norktans, B. (1981) Arch. Microbiol. 128, 267-270.
[4] Shaw, D.J. (1969) Electrophoresis. Academic Press, London.
[5] Daniels, S.L. (1971) Proceedings of the 28th General Meeting of the Society for Industrial Microbiology, Fort Collins,
CO, August 29-September4: Developments in Industrial
[6]
[7]
[8]
[9]
Microbiology, Vol. 13, pp. 211-253. American Institute of
Biological Sciences, Washington, DC.
Wood, J.M. (1980) in Microbial Adhesion to Surfaces
(Berkeley, R.C.W., Lynch, J.M., Melling, J., Rutter, P.R.
and Vincent, B., Eds.), pp. 163-185, Ellis Horwoqd,
Chicbester.
Daniels, S.L. and Kempe, L.L. (1966) Chem Eng. Progr.
Symp. Ser. 62, 142-148.
Neihof, R.A. and Loeb, G.I. (1972) Limnol. Oceanogr. 17,
7-16.
Marshall, K.C. and Cruickshank, R.H. (1973) Arch. Microbiol. 91, 29-40.