FEMS MicrobiologyLetters 9 (1980) 29-33
CopyrightFederation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
A D H E R E N C E O F B A C T E R I A TO H Y D R O C A R B O N S : A SIMPLE M E T H O D F O R
MEASURING CELL-SURFACE HYDROPHOBICITY
M. ROSENBERG, D. GUTNICK and E. ROSENBERG
Department of Microbiology, George S. WiseFaculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel
Received 20 May 1980
Accepted 20 June 1980
1. Introduction
The hydrophobic nature of the outermost surface
of various microbial cells has been implicated in such
biological phenomena as interactions between bac-
teria and phagocytes [1], attachment of bacteria to
host tissue [2,3], adherence of bacteria to non-
wettable solid surfaces [4,5], partitioning of bacteria
at liquid : liquid [6] and liquid : air [7,8] interfaces,
and the ability of microbial cells to grow on hydro-
carbons through direct contact with the immiscible
substrate [9-12].
A number of methods for studying hydrophobic
interactions of cells have been reported in the litera-
ture. These include binding of hydrocarbon and fatty
acids to cells and cell components [12,13], measure-
ment of the force required to remove hydrocarbon-
bound cells [9], partitioning of bacteria in aqueous
polymer two-phase systems [ 14,15], hydrophobic
interaction chromatography [3,15] and contact angle
measurements of dried cell layers [1,16]. No single
method adequately describes cell-surface hydro-
phobicity since (1) the experimental conditions em-
ployed influence the observed hydrophobic inter-
actions to some degree and (2) various non-hydro-
phobic effects often interfere.
Interest in the mechanism which enables direct
contact between hydrocarbon-degrading cells and
their water-insoluble alkane substrates has led us to
develop a rapid quantitative assay for the hydro-
phobic interaction of ceils with liquid hydrocarbons.
method is based on the degree of adherence of
cells to various liquid hydrocarbons following a brief
period of mixing. The present report describes this
29
technique and its application in measuring the surface
hydrophobicity of various bacterial cells.
2. Materials and Methods
2.1. Bacteria
The following test bacteria were used: Acineto-
bacter calcoaceticus strain RAG-1 (ATCC 31012) was
isolated previously in this laboratory [ 17,18] ; A. cal-
coaceticus strain BD 413 tryp E 27 was kindly pro-
vided by Dr. E. Juni. Escherichia cell B ilvA thy,
E. coli K-12 CSH 57, E. coil J-5, Bacfflus subtilis 168
and Enterobacter aerogenes CDC 659]66 were kindly
provided by Dr. E.Z. Ron. Micrococcus lysodeikticus
ATCC 4698 was kindly pr6vided by Dr. I. Friedberg.
Locally isolated strains of Staphylococcus aureus,
Staphylococcus albus and Serratia marcescens were
kindly provided by Ruth Zack. Pseudomonas aeru-
ginosa PAS 279 was kindly provided by Dr. J. Sha-
piro. The test bacteria were grown at 30C with shak-
ing in nutrient broth. Unless otherwise stated, bac-
teria were harvested at earlY logarithmic growth
phase, washed twice and resuspended in PUM buffer,
pH 7.1 : 22.2 g K2I-IPO4 3H20, 7.26 g KH2PO4,
1.8 g urea, 0.2 g MgSO4 7H20 and distilled water to
i 000 ml.
2.2. Assay procedure
To round-bottom test tubes (10 mm diameter),
containing 1.2 ml of washed cells suspended in PUM
buffer, were added various volumes of test hydrocar-
30
bon (n-hexadecane, n-octane or p-xylene). Following
10 min preincubation at 30C, the mixtures were agi-
tated uniformly on a Thermolyne Maxi Mix (Sybron)
for 120 s. After allowing 15 min for the hydrocarbon
phase to rise completely, the aqueous phase was care-
fully removed with a Pasteur pipette and transferred
to a 1 ml cuvette. Light absorbance was determined
at 400 nm, using a Gilford Model 240 spectropho-
tometer.
3. Results
The basic experiment used to measure bacterial
hydrophobicity is shown qualitatively in Fig. 1. Hexa-
decane was layered onto a turbid aqueous suspension
of Acinetobacter RAG-1 (Fig. 1, left tube) and then
mixed for 120 s; on standing a clear bottom layer and
a "creamy" upper layer formed (Fig. 1, middle tube).
Microscopic examination of the upper layer revealed
an oil-in-water emulsion consisting of hexadecane
droplets covered with patches of bacteria (Fig. 2).
Decrease in absorbance of the lower aqueous phase
was used as a measure of cell surface hydrophobicity.
Fig. 1. Adherence of Acinetobacter RAG-1 to hydrocarbon.
Hexadecane was added to an aqueous suspension of Acineto-
bacter RAG-1 (left). After mixing for 120 s and allowing to
stand, adherent cells rose with the hydrocarbon, forming a
"creamy" upper layer and a clear aqueous phase (middle).
Addition of isopropanol to a final concentration of 5% (v/v)
results in breakage of the emulsion and release of cells back
into the aqueous phase (right).
The cell-stabilized emulsions did not break even after
several days; however, addition of 5% (v/v) iso-
propranol brought about an immediate coalescence of
droplets and release of cells into the aqueous phase
(Fig. 1, right tube).
Results illustrating the adherence of various bac-
teria to the test hydrocarbons are presented in Figs.
3 - 6 . Fig. 3 compares the affinity ofE. coli with
those of two Acinetobacter strains. E. coli B showed
no significant affinity towards hexadecane or octane
and little or no affinity towards xylene. Similar
results (not presented) were obtained with M. lyso-
deikticus, E. aerogenes, B. subtilis and P. aeruginosa.
While Acinetobacter RAG-1 showed high affinity
towards all three hydrocarbons, Acinetobacter BD
exhibited higher affinity for octane and xylene than
for hexadecane. More than 97% of the RAG-1 and
over 99% of the BD strain cells were removed from
the aqueous phase by 0.2 ml octane.
The affinities of two species of Staphylococci
towards the test hydrocarbons are presented in Fig. 4.
Although S. albus did not adhere to hexadecane or
xylene, S. aureus exhibited a high affinity towards all
the test hydrocarbons.
Although S. marcescens grown to early exponen-
tial phase showed relatively low affinity towards
hexadecane and xylene and none towards octane,
pink-pigmented early stationary phase cells adhered
strongly to all three hydrocarbons (Fig. 5).
Loss of oligosaccharide components on the outer
surface ofE. coli rough mutant J-5 [19] was accom-
panied by a significant increase in its affinity towards
Fig. 2. Phase micrograph showing RAG-1 cells adhering to
hexadecane droplet following mixing. 825X.
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Figs. 3-6. Aff'mityof bacteria towards hydrocarbon as a function of hydrocarbon volume. Aqueous bacterial suspensions were
mixed with varying volumes of hexadeeane, octane and xylene as described in Materials and Methods. Results are expressed as
percentage of the initial absorbanee (A 400) of the aqueous suspension as a function of hydrocarbon volume. Initial absorbance is
shown in parentheses.

each of the three test hydrocarbons, as compared
with E. coli K-12 (Fig. 6).
4. Discussion
A simple quantitative method has been described
for studying the outer cell surface of bacteria, based
on the affinity of these cells for liquid hydrocarbons.
Large differences in the aftrmities of various bacteria
for hydrocarbons have been demonstrated using this
technique.
Previous observations have suggested that the abil-
ity to adhere to bulk hydrocarbon is a characteristic
of hydrocarbon-degrading microorganisms [ 11 ]. We
have shown that S. aureus and early stationary phase
S. marcescens cells adhere to a variety of fiquid hy-
drocarbons despite their inability to degrade them.
Thus, the ability of bacterial cells to adhere to hydro-
carbon is not restricted to hydrocarbon-degrading
bacteria. Moreover, both Acinetobacter strains
adhered strongly to octane and xylene, hydrocarbons
which they are incapable of metabolizing. This indi-
cates that the adherence of these hydrocarbon-
degrading microorganisms is not limited to metabo-
lizable hydrocarbons, and suggests that direct contact
between Acinetobacter cells and bulk hydrocarbon is
due to general hydrophobic interactions rather than
specific recognition of the substrate. P. aeruginosa
PAS 279 did not adhere significantly to the test hy-
drocarbons under the conditions studied, despite its
ability to grow on hexadecane. This result suggests
that affmity for hydrocarbon may vary among hydro-
carbon-degrading bacteria. Further studies on the
relationship between adherence to hydrocarbon and
growth of hydrocarbon-degrading bacteria are under-
way.
The large increase in hydrophobicity observed in
S. marcescens with increasing age of the ceils agrees
with previous reports [8,13]. The small, but signifi-
cant increase in the affmity of the rough mutant
E. coli J-5 as compared with that ofE. coli K-12 indi-
cates the increased hydrophobicity of the rough mu-
tant, presumably due to the increased exposure of
inner core regions of the LPS layer. Similar results
have been obtained with rough mutants of Salmonella
typhimurium [1,14].
The method described here may prove useful in
enabling the separation of certain cell mixtures, and
in separating hydrophobic cell components. In addi-
tion, this technique may provide a means of enriching
for and selecting mutants which are altered in their

surface hydrophobicity. A search for such mutants is
currently in progress, with a view towards investigat-
ing the relationship between the metabolism o f hy-
drophobic substrates and modifications in cell surface
hydrophobicity.
Acknowledgements
We thank Z.L. Zosim, L. Goldstein and E.A. Bayer
for stimulating discussions and constructive criticism.
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