Journal of Microbiological Methods 60 (2005) 225 233

www.elsevier.com/locate/jmicmeth

Measurement of bacterial adhesionin vitro evaluation

of different methods
Satu Vesterlunda,*, Johanna Palttab, Matti Karpb, Arthur C. Ouwehanda
a

Department of Biochemistry and Food Chemistry, University of Turku, Itainen Pitkakatu 4A, 20014 Turku, Finland
b
Department of Biotechnology, University of Turku, Tykistokatu 6, 20014 Turku, Finland
Received 27 September 2004; accepted 27 September 2004

Abstract
The adhesion of bacteria to host tissue is the first step in pathogenesis. Similarly, bacterial adhesion to inanimate surfaces is
the first step in formation of biofilmsa real problem in industrial processes and medical devices. Various agents capable of
blocking the adhesion of bacteria to surfaces have been identified, such as probiotics, which are supposed to prevent the
adhesion of pathogenic bacteria to the intestinal mucosa. Although measurement of bacterial adhesion is important itself,
especially when agents used to prevent adhesion are developed, a relative small number of techniques can be used in the
measurement of adhesion. These techniques are not well validated and there is lack of studies where those methods are
compared to each other. Here we have compared different commonly used methods to measure adhesion of bacteria; radioactive
labelling, fluorescence tagging, and staining of bacteria. The methods were used to measure the adhesion of Escherichia coli
and Salmonella enterica serovar Typhimurium to intestinal mucus. Moreover, selected probiotic strains were used to study
whether probiotics or the adhesion method used affected the results. As a result, we show that the best reproducibility and
sensitivity were obtained using radioactive labelling. With other methods, the sensitivity was too low due to poorly adhering
bacteria and low signal-to-background ratio.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Adhesion; Crystal violet; DAPI; EYFP; Fluorescence; GFP; Probiotic; Radioactively labelled

1. Introduction
An often necessary step in the infection process is
the adhesion of pathogenic bacteria to host tissues
* Corresponding author. Tel.: +358 2 333 6823; fax: +358 2
333 6860.
E-mail address: satu.vesterlund@utu.fi (S. Vesterlund).
0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2004.09.013

(Finlay and Falkow, 1997). Also the formation of

biofilms in industrial processes is mostly an unfavourable event mediated through the adhesion of
bacteria to various surfaces. A considerable amount
of research has been done to understand how bacteria
adhere to surfaces. Similarly different agents capable
of blocking the adhesion of bacteria to certain
surfaces have been developed. Oral probiotics are

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S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233

such agents that can interfere with the adhesion of

other microbes and have been defined as bliving
microorganisms, which, upon ingestion in certain
numbers, exert health benefits beyond inherent basic
nutritionQ (Guarner and Schaafsma, 1998). Although
adhesion is the cornerstone of pathogenesis, a relative
small number of techniques can be used to measure
the adhesion. Conventional methods used to enumerate the adherent bacteria are based on plating or
microscopic counting. Plating is often used when the
amount of bacteria adhered to eukaryotic cells
(Bermudez et al., 1994) or inanimate surfaces
(Gristina et al., 1989; Sheehan et al., 2004) is
detected. However, this method is laborious and
requires that bacteria are released first and that
bacteria remain culturable after the release process.
Microscopic evaluation can be used after fixation and
Gram staining of the bacteria (Tuomola and Salminen,
1998), but the method is equally laborious as plating.
When the amount of released bacteria is sufficient for
turbidometric analysis, spectrophotometry can be
used (Styriak et al., 1999), but the sensitivity and
accuracy are maybe less compared to plating or
microscopic enumeration. These methods cannot be
used when adhesion of one bacterial strain is studied
in an environment where other bacteria are present. In
order to distinguish bacteria in a mixed population,
radiolabels (Ahearn et al., 2000; Jin et al., 1998;
Tuomola et al., 1999), fluorochromes (Bosch et al.,
2003; Drudy et al., 2001), or bacteria-specific antibodies (Sanchez et al., 1993) can be used. Radiolabels
are regarded as undesirable due to safety and cost
concerns. Fluorochromes are used to replace radiolabels, but they may alter the surface properties of
bacteria or affect the viability of bacteria (Fuller et al.,
2000). Bacteria-specific antibodies can be used, but
the availability can cause problems as well as crossreactions.
There is a lack of studies where different adhesion
methods are compared to each other. Since detection
of bacterial adhesion is important (e.g., in the
development of probiotics), much more studies
should be done where different methods are evaluated. In this study, we have compared commonly
used methods to measure the adhesion of bacteria.
The methods were based on radiolabelling, fluorescent tagging (measured by microscopy or fluorescence), and staining (with crystal violet and DAPI).

These methods are used to detect the adhesion of

Escherichia coli and Salmonella enterica serovar
Typhimurium bacteria to intestinal mucus. Moreover,
we studied whether the selected commercial probiotic strains, Lactobacillus casei Shirota (hereafter
LcS) and Lactobacillus rhamnosus GG (hereafter
LGG) affected the adhesion of pathogenic bacteria,
and, more importantly, whether the adhesion method
used affected the results.

2. Materials and methods

2.1. Plasmid constructs of transformed strains
Plasmids containing genes for fluorescent proteins
(GFPmut2 and EYFP; Clontech, Palo Alto, CA) were
transformed into E. coli and S. enterica serovar
Typhimurium, respectively, by electroporation (Dower
et al., 1988) and selection for ampicillin (100 Ag ml 1)
resistance.
2.2. Bacterial strains and culture conditions
Bacterial strains used were E. coli MC1061,
GFPmut2-tagged variant from E. coli MC1061, S.
enterica serovar Typhimurium ATCC 14028 and
EYFP-tagged variant of this strain. Probiotic strains
used were lactic acid bacteria (LAB): L. casei
Shirota (isolated from a YakultR product; LcS) and
L. rhamnosus GG (ATCC 53103; LGG). All strains
were stored at 86 8C in 40% glycerol. E. coli and
S. enterica serovar Typhimurium were plated first
onto LuriaBertani (LB; yeast extract and tryptone
were purchased from Pronadisa, Madrid, Spain)
plates supplemented with 100 Ag ml 1 ampicillin.
Plates were grown for 16 h at 37 8C and stored for
not more than 2 weeks in 4 8C. For culturing, one
colony was inoculated into 5 ml of LB broth
supplemented with 100 Ag ml 1 ampicillin, and the
culture was grown for 16 h without agitation at 30 8C
to reach stationary growth phase. LcS and LGG were
inoculated directly from glycerol stocks as a 0.5%
inoculum into de Man, Rogosa, and Sharpe (MRS)
broth (Oxoid, Basingstoke, UK). Bacteria were
grown for 18 20 h without agitation at 37 8C under
anaerobic conditions in order to reach the late
logarithmic growth phase. Bacteria were harvested

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233

by centrifugation (1500g, 7 min) and washed twice

with phosphate-buffered saline (PBS; pH 7.2). The
optical density of bacterial suspensions at 600 nm
was adjusted with PBS to 0.5F0.02, giving approximately 4108 CFU ml 1 for E. coli and S. enterica
serovar Typhimurium strains and 1 2108 CFU
ml 1 for LcS and LGG.
2.3. Human intestinal mucus
Human intestinal tissue was used as a source of
mucus. The use of resected human intestinal tissue
was approved by the joint ethical committee of the
University of Turku and Turku University Central
Hospital and informed written consent was obtained
from the patients. The mucus was isolated from the
healthy part of tissue obtained from a patient with
diverticulitis. In short, resected material was collected on ice within 20 min and processed immediately by washing gently with PBS containing 0.01%
gelatin. Mucus was collected into a small amount of
HEPESHanks buffer (10 mmol l 1 HEPES; pH
7.4) by gently scraping with a rubber spatula and
centrifuged (13,000g, 10 min) in order to remove
cell debris and bacteria. After measurement of the
protein content, mucus was stored at 20 8C. The
same stock of mucus was used in all experiments in
order to avoid the possible effect of variations in the
mucus on adhesion. In adhesion assays, mucus was
diluted to a protein concentration of 0.5 mg ml 1
with HEPESHanks and 100 or 50 Al of this
solution was immobilized passively into microtiter
plate wells (Maxisorp; Nunc, Denmark) or into
microscope slides, respectively, by overnight incubation at 4 8C.
2.4. In vitro adhesion assays
2.4.1. Adhesion of radioactively labelled bacteria
When the original and fluorescent strains of E.
coli and S. enterica serovar Typhimurium were
inoculated as described above, 10 Al ml 1 [5V-3H]
thymidine (16.7 Ci mmol 1) was added to the
cultures to metabolically radiolabel the bacteria.
After growth, washing, and adjustment of optical
density at 600 nm to 0.5, bacteria were added as a
volume of 100 Al into microtiter plate wells coated
with human intestinal mucus. Bacteria were allowed

227

to adhere at 37 8C for 1 h and the wells were

washed three times with 250 Al of HEPESHanks
buffer to remove the nonadherent bacteria. The
bacteria bound to mucus were released and lysed
with 1% SDS 0.1 M NaOH by incubation at 60 8C.
The radioactivity of the suspension was measured by
liquid scintillation. Three parallel wells were used in
each experiment. The adhesion ratio (%) of bacteria
was calculated by comparing the radioactivity of the
adhered bacteria to the radioactivity of the added
bacteria.
2.4.2. Adhesion of fluorescent-tagged bacteria
measurement by fluorometer
After adjustment of optical density at 600 nm to
0.5, GFPmut2/E. coli and EYFP/S. enterica serovar
Typhimurium were added as a volume of 100 Al into
microtiter plate wells coated with mucus. After incubation and washing as described in Section 2.4.1, the
wells were covered with 100 Al of HEPESHanks
buffer to prevent drying of the bacteria. Fluorescence
of the bacteria was measured by using a Victor2 1420
Multilabel counter (PerkinElmer, Turku, Finland).
The filters used were 485 nm filter for excitation
and 535 nm filter for emission. The sensitivity of the
measurement was increased by measuring 12 different
points (the diameter of one point was 4 mm) from one
well. Fluorescence of immobilized mucus covered
with 100 Al of HEPESHanks buffer was used as a
background in measurements, and it was reduced
from the fluorescence of the samples. Three parallel
wells were used in each experiment. The adhesion
ratio (%) of bacteria was calculated by comparing the
fluorescence of the adhered bacteria to the fluorescence of the added bacteria.
2.4.3. Adhesion of fluorescent-tagged bacteria
measurement by microscopic counting
In microscopic analysis, the fluorescent E. coli
MC1061 and S. enterica serovar Typhimurium were
added as a volume of 50 Al into microscope slides
covered with mucus (Krovacek et al., 1987). Microscope slides were incubated in humidified chamber
at 37 8C for 1 h and the nonadherent bacteria were
washed away by dipping the slides into 0.9% NaCl
solution. The slides were covered with a coverslip
and stored in humidified chamber until enumeration
of adherent bacteria with epifluorescence microscope

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(Olympus BX51, Japan). Adhered bacteria in 20

randomly selected fields were enumerated.
2.4.4. Detection of bacterial adhesion with crystal
violet
The crystal violet method was modified after
Styriak et al. (1999). In short, E. coli and S.
enterica serovar Typhimurium (nonfluorescent and
fluorescent) bacteria were added as a volume of 100
Al into microtiter plate wells coated with 150 Al of
human intestinal mucus. The bigger volume of
mucus compared to the volume of added bacteria
was used to avoid contact of the stain with the
polystyrene. Bacteria were adhered at 37 8C for 1 h
and the nonadherent bacteria were removed by
washing the wells three times with 250 Al of
PBS. The adherent bacteria were fixed at 60 8C
for 20 min and stained with crystal violet (100 Al
well 1, 0.1% solution) for 45 min. Wells were
subsequently washed five times with PBS to remove
excess stain. The stain bound to bacteria was
released by adding 100 Al of citrate buffer (20
mmol l 1; pH 4.3). After 45-min incubation at room
temperature, the absorbance values at 570 nm were
determined by using Victor2 1420 Multilabel counter (PerkinElmer). Stained mucus without added
bacteria was used as negative control and the
absorbance value of this negative control was
subtracted from the absorbance value of the
samples. Four parallel wells were used in two
independent experiments.
2.4.5. Detection of bacterial adhesion with 4V,6diamidino-2-phenylindole (DAPI)
PBS-washed E. coli and S. enterica serovar
Typhimurium bacteria were stained with DAPI by
adding the stain as a final concentration of 0.2 Ag
ml 1. Bacteria were incubated for 30 min with mild
shaking at room temperature and washed three times
with PBS. Adhesion assay was done as described in
Sections 2.4.1 and 2.4.2. Then, wells were washed
with 250 Al of HEPESHanks buffer and the
fluorescence from the wells was determined by using
Victor2 1420 Multilabel counter (PerkinElmer). The
filters used were 355 nm filter for excitation and 460
nm filter for emission. Sensitivity of the measurement
was increased, as was done in the fluorescence study,
by measuring 12 different points (the diameter of one

point was 4 mm) from one well. The staining

efficiency of the bacteria was determined microscopically (Section 2.4.3). Four parallel wells were used in
three independent experiments. The adhesion ratio
was calculated as described in Section 2.4.2.
2.5. Effect of LAB on adhesion ability of pathogens
The effect of LcS and LGG on the adhesion
ability of E. coli MC1061 and S. enterica serovar
Typhimurium was assessed in exclusion (LAB was
incubated first with the mucus, washed away, and
followed by incubation with pathogenic bacteria),
competition (bacteria were incubated simultaneously), and displacement (pathogenic bacteria were
incubated first with the mucus, washed away, and
followed by incubation with LAB) assays. The effect
of LAB on the adhesion of E. coli and S. enterica
serovar Typhimurium was assessed with different
methods (Sections 2.4.1, 2.4.2, and 2.4.3) in order to
study whether the method used had effects on the
results.
2.6. Statistical analysis
Results shown from Sections 2.4.1, 2.4.2, and 2.4.3
are the averageFstandard deviation (S.D.) of four
independent experiments. Students t test was used to
determine the significant difference ( Pb0.05) between
the samples.

3. Results
3.1. Sensitivity of the methods
Sensitivity of the methods was determined by
measuring the signal obtained from a single bacterium. In radiolabelling, the lowest amount of
detectable bacteria after background subtraction was
2.7103 CFU for both GFPmut2/E.coli MC1061 and
EYFP/S. enterica serovar Typhimurium (Fig. 1). In
the fluorescence method, the lowest detectable signal
after background subtraction was obtained from
6.4104 CFU of GFPmut2/E. coli and 1.1105
CFU of EYFP/S. enterica serovar Typhimurium
bacteria (Fig. 2). Staining with crystal violet (Section
2.4.4) was not a sensitive-enough method to detect

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233

229

Fig. 1. Linear relationship between radioactivity and CFU of GFPmut2/E. coli and EYFP/S. typhimurium bacteria. Results shown are
averageFS.D. of three samples.

low levels of adherent bacteria as the signal was not

different from the background; absorbance values at
570 nm were between 0 and 0.05 in two independent
experiments. Similarly with DAPI staining (Section
2.4.5), the fluorescence obtained from the sample was
the same or close to the background. This was the
reason for high S.D. values in the experiments;
adhesion of E. coli was 2.80F3.04% and S. enterica
serovar Typhimurium was 0.41F0.72%.
3.2. Effect of LAB on adhesion ability of pathogens
When radiolabelling (Section 2.4.1) was used, the
adhesion percentages of E. coli, GFPmut2/E. coli, S.

enterica serovar Typhimurium, and EYFP/S. enterica

serovar Typhimurium bacteria were 0.70, 0.60, 0.50,
and 0.85, respectively (Table 1). Displacement with
LcS significantly reduced the binding (%) of E. coli
and S. enterica serovar Typhimurium to 0.41
( P=0.006) and 0.34 ( P=0.013), respectively. Similarly, displacement with LGG significantly reduced
the binding (%) of E. coli, S. enterica serovar
Typhimurium, and EYFP/S. enterica serovar Typhimurium to 0.49 ( P=0.041), 0.27 ( P=0.020), and 0.27
( P=0.032), respectively. Also with other bacteria, LcS
and LGG caused a trend for lowered adhesion in
displacement, but this did not reach statistical
significance: LcS and GFPmut2/E. coli ( P=0.310),

Fig. 2. Linear relationship between fluorescence and CFU of GFPmut2/E. coli and EYFP/S. typhimurium bacteria. Results shown are
averageFS.D. of three samples.

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Table 1
Adhesion assay using radioactively labelled bacteriaeffect of L. casei Shirota (LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens
Assay

E. coli

GFPmut2/
E. coli

S. typhimurium

EYFP/
S. typhimurium

Alone
Exclusion by LcS
Exclusion by LGG
Competition by LcS
Competition by LGG
Displacement by LcS
Displacement by LGG

0.70F0.19
0.47F0.09
0.78F0.52
0.44F0.12
0.57F0.13
0.41F0.15a
0.49F0.17a

0.60F0.17
0.45F0.05
0.62F0.12
0.70F0.45
0.50F0.16
0.47F0.06
0.40F0.07

0.50F0.18
0.71F0.48
0.44F0.11
0.74F0.32
1.69F1.42
0.34F0.18a
0.27F0.10a

0.85F0.32
0.68F0.09
0.64F0.29
0.67F0.26
0.95F0.22
0.62F0.37
0.27F0.09a

Adhesion (%); meanFS.D. of four independent experiments.

a
Significantly lower than bacteria incubated alone with the mucus ( Pb0.05).

LGG and GFPmut2/E. coli ( P=0.064), as well as LcS

and EYFP/S. enterica serovar Typhimurium ( P=
0.398). It was also important for the use of fluorescent
bacteria that the fluorescent phenotype did not affect
the adhesion of bacteria (Table 1).
Use of fluorescent bacteria and fluorometer (Section 2.4.2) gave lower binding (%) for GFPmut2/E.
coli and EYFP/S. enterica serovar Typhimurium (0.14
and 0.07, respectively) when compared to radiolabelling (Table 2). The S.D. values were high due
to relative low reproducibility, use of poorly adherent
bacteria, and probably also light scattering and
autofluorescence of the mucus. Thus, LcS and LGG
did not statistically affect the adhesion ability of the
pathogens.
In order to compare the microscopic method
(Section 2.4.3) to other methods, the results from
Sections 2.4.1, 2.4.2, and 2.4.3 were represented as a
number of adherent bacteria per area of the mucus
(mm2). With fluorescent bacteria and fluorometer, the

Table 2
Adhesion assay using fluorescent-tagged bacteriameasurement by
fluorometer and effect of L. casei Shirota (LcS) and L. rhamnosus
GG (LGG) on adhesion of pathogens
Assay

GFPmut2/
E. coli

EYFP/
S. typhimurium

Alone
Exclusion by LcS
Exclusion by LGG
Competition by LcS
Competition by LGG
Displacement by LcS
Displacement by LGG

0.14F0.11
0.09F0.10
0.12F0.12
0.21F0.06
0.20F0.07
0.14F0.03
0.19F0.05

0.07F0.02
0.07F0.07
0.16F0.12
0.06F0.05
0.07F0.03
0.05F0.04
0.07F0.03

Adhesion (%); meanFS.D. of four independent experiments.

number of adherent bacteria was almost always

significantly lower when compared to the radiolabelling method (Tables 3 and 4). In the sample where
LcS and GFPmut2/E. coli were incubated together
(competition), a trend for a reduction in adhesion was
observed ( P=0.076). Using fluorescent bacteria and
microscopy, the number of bacteria observed was
often higher when compared to radiolabelling,
although statistical significance was obtained only in
competition of YFP/S. enterica serovar Typhimurium
by LGG ( P=0.007; Tables 3 and 4). Similarly, when
microscopy was compared to fluorometry, significantly higher numbers of bacteria were obtained with
microscopy: exclusion of GFPmut2/E. coli by LGG

Table 3
Adhesion of GFPmut2/E. coli per surface area (mm2)
Assay

Radiolabelled Fluorescent Fluorescent

fluorometry microscopy

Alone
Exclusion by LcS
Exclusion by LGG
Competition by LcS
Competition by LGG
Displacement by LcS
Displacement by LGG

6.26F1.72
4.65F0.57
6.48F1.26
7.23F4.69
5.24F1.70
4.88F0.57
4.11F0.68

1.46F1.13a 22.00F24.03
0.89F1.05b 9.63F10.93
1.22F1.29a 4.75F2.26c
2.16F0.65
3.50F2.16
2.08F0.74a 39.88F24.03
1.47F0.27b 10.50F6.72c
2.01F0.55a 8.38F7.94

Measurement with different methods and effect of L. casei Shirota

(LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens.
Results shown are 103 of adherent bacteria per square millimeter;
meanFS.D. of four independent experiments.
a
Significantly lower than the result obtained with radiolabelling ( Pb0.05).
b
Significantly lower than the result obtained with radiolabelling ( Pb0.001).
c
Significantly higher than the result obtained with fluorometry
( Pb0.05).

S. Vesterlund et al. / Journal of Microbiological Methods 60 (2005) 225233

Table 4
Adhesion of EYFP/S. typhimurium per surface area (mm2)
Assay

Radiolabelled Fluorescent Fluorescent

fluorometry microscopy

Alone
Exclusion by LcS
Exclusion by LGG
Competition by LcS
Competition by LGG
Displacement by LcS
Displacement by LGG

8.78F3.36
7.09F0.96
6.62F2.98
7.00F2.74
9.89F2.34
6.41F3.80
2.81F0.90

0.71F0.25a
0.73F0.71d
1.61F1.27a
0.66F0.51a
0.68F0.31d
0.51F0.40a
0.68F0.30a

23.88F10.21b,c
15.00F6.65b
19.25F11.87b
20.63F16.19b
30.13F9.78c,e
14.75F12.84
26.88F29.71

Measurement with different methods and effect of L. casei Shirota

(LcS) and L. rhamnosus GG (LGG) on adhesion of pathogens.
Results shown are 103 of adherent bacteria per square millimeter;
meanFS.D. of four independent experiments.
a
Significantly lower than the result obtained with radiolabelling ( Pb0.05).
b
Significantly higher than the result obtained with fluorometry
( Pb0.05).
c
Significantly higher than the result obtained with radiolabelling ( Pb0.05).
d
Significantly lower than the result obtained with radiolabelling ( Pb0.001).
e
Significantly higher than the result obtained with fluorometry
( Pb0.001).

( P=0.035), displacement of GFPmut2/E. coli by LcS

( P=0.036), exclusion of EYFP/S. typhimurium by
LcS ( P=0.005) and LGG ( P=0.025), as well as
competition of YFP/S. typhimurium by LcS
( P=0.049) and LGG ( P=0.001) (Tables 3 and 4).

4. Discussion
Bacterial adhesion is one of the main concerns in
the areas of medicine, industry and environment. In
many cases, bacterial adhesion is unwanted as it can
lead to infection or interruption of the industrial
processes. However, bacterial adhesion can also be an
advantage as in the case when probiotics are used to
promote intestinal health (Mattila-Sandholm et al.,
1999). As bacterial adhesion is involved in many
sectors of life and health, the development of methods
to measure adhesion is an important area.
The adhesion method used is often selected on the
basis of what people are accustomed to use. As
adhesion of bacteria is thought to be a complex
interplay between bacteria and surface, the adhesion
method used could affect the results. The initial step in
the adhesion process is mainly a physicochemical

231

process, based on nonspecific interactions (i.e.,

repulsive electrostatic and attractive van der Waals
interactions) (van Loosdrecht et al., 1990). This kind
of adhesion can be reversible and, therefore, the
number of washings used in adhesion assays should
remain constant between experiments. Initial adhesion
is followed by firm attachment where adhesins on the
bacterial cell surface recognize receptors on the target
surface (Miron et al., 2001). As the stain used could
change the surface properties of the bacterial cell, for
example, by affecting to the hydrophobicity of the cell
(Olofsson et al., 1998), the stain may affect the
adhesion of bacteria. Adhesion of microbes has also
been found to be increased during exponential growth,
possibly as a result of increased cell wall hydrophobicity (van Loosdrecht et al., 1990). Thus, when
adhesion properties of different strains are compared,
the growth phase of microbes should be the same in
order to reach comparable results (Blum et al., 1999).
A limited number of methods used to measure
bacterial adhesion are commonly used. The conventional methods used are enumeration by plating or
microscopy. However, plating is laborious and
insensitive as it requires selective media, and the
method does not detect cells that are viable but not
culturable (Rahman et al., 1994; Steinert et al., 1997)
or which died during the release process. Similarly
enumeration by microscopy after staining of the
sample is laborious as it requires counting of many
fields and may also be prone to observer error.
Furthermore, these methods cannot be used when
adhesion of bacteria is studied in a mixed population. In these kind of applications, fluorochromes can
be used. Although these are usually developed to
stain eukaryotic cells, many fluorochromes are also
suitable for prokaryotic use. Use of genetically
modified, fluorescent-tagged bacteria is preferred
over fluorescent stains as they make the experiments
shorter, do not stain other bacteria when mixed
populations are studied, and, above all, the signal
propagates with the dividing bacteria, avoiding the
dilution of the signal. Green fluorescent protein
(GFP) is the most widely used fluorescent marker.
The popularity of the GFP is due to its properties:
heat stability (up to 65 8C), pH stability (pH 7 to
11), resistance to denaturants and proteases, and no
requirement of added cofactors for fluorescence
(Aspiras et al., 2000), as well as its expression in a

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wide variety of bacterial species (Valdivia et al.,

1998). However, certain difficulties can occur when
wild-type GFP is used. The fluorescence intensity or
folding can be too low, or protein is found in
nonfluorescent inclusion bodies. Thus, the properties
of GFP have been improved by mutagenesis in order
to overcome these problems. Here we used GFPmut2
variant, which has been shown to fluoresce at
approximately 100-fold higher intensity and to be
more soluble compared to wild type (Cormack et al.,
1996). Similarly, for Salmonella, another improved
GFP variant, EYFP, was used. Both fluorochromes
were bright under microscope and showed 100%
labelling, and no photobleaching was observed
during experiments. The fluorescent phenotype was
not observed to affect the growth of the bacteria,
indicating that the expression levels did not pose any
metabolic burden on bacteria (results not shown).
However, the sensitivity of the method where
fluorescence was detected by fluorometry did not
reach the sensitivity that was obtained with radiolabelled bacteria. The signal obtained from radiolabelled bacteria was approximately two to four log
units higher than the signal obtained with fluorometery (Figs. 1 and 2). Although the fluorescence
intensity of bacteria could be improved by increasing
the gene copy number or using a stronger promoter,
the higher level of fluorescent protein may interfere
with the adhesion properties of bacteria (Wendland
and Bumann, 2002). Longer maturation of the
protein was not a solution for low fluorescence
either; when bacteria were kept overnight at 4 8C,
the fluorescence was at the same level, indicating
proper maturation of the protein. When the fluorescent bacteria were enumerated by microscope, the
S.D. was high due to low adhesion of bacteria (010
bacteria per field). Thus, the use of fluorescenttagged bacteria is probably not a suitable method
when poorly adherent bacteria (b1%) are studied.
Similarly, the use of common stains, fluorescent
DAPI, and crystal violet did not work with poorly
adherent bacteria. In these methods, also the use of
mixed bacterial population (i.e., here the intact
bacterial microbiota present in the mucus) made
the methods more insensitive as the microbiota was
probably also stained.
In summary, the use of radioactive labels in
bacterial adhesion assays offers the best reproduci-

bility and sensitivity when poorly adherent bacteria

(b1%) are studied. The use of fluorescent-tagged
bacteria enables also easy and reproducible enumeration of adherent bacteria, but the sensitivity is low
for poorly adherent bacteria. This is due to high
signal-to-background noise especially when the
adhesion surface is autofluorescent. Because with
radioactive labels the safety issues are the main
disadvantage, and considering the advantages and
potential of fluorescent-tagged bacteria, more studies
are needed to increase the sensitivity of bacterial
adhesion methods based on the use of such tagged
bacteria.

Acknowledgements
Financial support was obtained from the Academy
of Finland (grant no. 53758), the Danisco Foundation,
and the Paulo Foundation.

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