Current Issues on Multidisciplinary Microscopy Research and Education 93

Microscopy techniques applied for monitoring the development

of aquatic biofilms
Jordi Morató*,1, Francesc Codony1, and Jordi Mas2
1
Lab. Health and Environmental Microbiology, Universitat Politècnica de Catalunya, E-08222-Terrassa
(Barcelona) Spain
2
Department of Genetics and Microbiology. Universitat Autònoma de Barcelona, E-08193-Bellaterra
(Barcelona) Spain

Using an experimental setup based on packed bed reactors (filled with glass beads) supplied with water
from a groundwater source, the suitability of different microscopic techniques for biofilm monitoring was
evaluated. Confocal microscopy is the most versatile, applicable and suitable technology for biofilm study
and even provides information about the main surface and height parameters. Nevertheless, the wide field
images obtained with the confocal perfilometer, combined with the super-long work distances of the ob-
jectives used, facilitate and improve the observation of non-flat surfaces. Moreover, the utilization of a
white light source does not affect the viability and the activity of microbial cells. Confocal perfilometry
can be the choice for a non-invasive monitoring of the microbial colonization of non-flat surfaces, such as
the glass beads. Other techniques such as the Environmental Scanning Electron Microscopy (ESEM),
cryoembedding and cryosectioning can be useful as complementary tools to help in the characterization of
the structure and architecture of mature biofilms.

Keywords biofilm monitoring, confocal microscopy, confocal perfilometer, fluorescence microscopy, vi-
ability stains, cryoembedding and cryosection

1. Introduction
Bacteria present in aquatic environments can be often found adhered to surfaces, forming biofilms [1].
These structures can be found in different degrees of complexity, ranging from single-cell layered con-
structions showing a high degree of patchiness, low cell numbers and limited presence of polymeric
compounds; to multilayered, well developed constructs constituted by several layers of cells embedded
in a complex polymeric matrix [2, 3]. When biofilm growth occurs associated to man made structures, a
number of problems have been reported [4, 5]: decreased heat transfer in heat exchangers, contamination
of fluids in food industry, colonization on medical implants and contact lenses, microbial corrosion of
conductions, etc., all resulting in increased hazards and often in straight economic losses directly or indi-
rectly associated with biofilms. Moreover, biofilms could act as promoters for regrowth of microbial
contaminants in water distribution systems [6].
Control of biofilm build-up is becoming therefore, of paramount importance in a large number of unre-
lated fields, and the development of simple and sensitive methods for biofilm monitoring is essential.
There is already a rather large choice of methodologies for biofilm monitoring (see [7, 8] for a review).
Nevertheless, most of the methodologies used require the destructive sampling of biofilms, and do not
provide information about their architecture, spatial structure and physiological heterogeneity. Moreover,
most viable bacteria cannot be cultured under conventional microbiologic methods [9].
The development of non-invasive and non-destructive techniques, such as confocal microscopy has
stimulated the research on microbial biofilms. With the aid of confocal scanning laser microscope
(CSLM) different optical sections –horizontal or vertical- of fully hydrated and intact live biofilms can
be performed [2]. Used in conjunction with different fluorescent molecular probes, the CSLM can pro-
vide information on cell morphology, metabolism and phylogeny, and at the same time on biofilm matrix
structure and architecture [1]. Different specific fluorochromes [10, 11] or antibodies coupled with FITC
*
Corresponding author: e-mail: morato@oo.upc.edu

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94 Jordi Morató et al.: Microscopy techniques applied for monitoring the development of aquatic biofilms

[12] can be used to stain most biofilms, and with the aid of fluorescence microscopy [13, 14] or flow
citometry [15], the total or viable cell populations can be quantified. The application of fluorochromes
can even be used to analyze the physiological heterogeneity of natural and intact biofilms, especially for
thin samples [14]. For thick biofilms it is possible to freeze the samples (cryoembedding) before their
staining, and after this, different thin sections of the entire biofilm can be performed with a cryotome
(cryosectioning), maintaining its structural integrity [16].
The advent of different molecular probes has enhanced the staggering versatility of confocal microscopy,
and has supposed a substantial impact on our knowledge of biofilm ecology. In this sense, fluorescent in
situ hybridization (FISH) with probes for 16S rRNA allows the possibility to identify the phylogeny of
each group of cells that constitute the biofilm and even to analyze its genetic diversity [17,18].
Using an experimental setup based on packed bed minireactors (filled with glass beads) supplied with
water from a groundwater source [19, 20], the suitability of different microscopic techniques for biofilm
monitoring was evaluated. These minireactors provide a compact, rugged, inexpensive and versatile
system to measure directly the extent of biofilm formation in water systems, by periodical removing
biofilm-attached glass beads for off-line biofilm analyses [19, 20]. With this system, biofilm develop-
ment and cell viability can be easily monitored, either in laboratory or in environmental conditions. The
experimental system constructed allows the sampling of undisturbed biofilms grown under realistic flow
conditions and to quantify the kinetics of biofilm development. Our study addressed the application of
several microscopic techniques to packed bed reactors for monitoring biofilm development under field
conditions.

2. Materials and Methods

2.1 Biofilm sampling and analyses

Sampling of attached biomass required periodic removal of individual reactors for off-line, both non-
destructive and destructive, biofilm analyses. Samples were collected once a week from the samplers
(packed-bed reactors) in the field to quantify the biofilm formation on glass beads after the exposition to
a groundwater source. Once a sample was taken, the biofilm was directly observed with different micro-
scopic techniques (see below). The samples were also analyzed for viable and culturable bacterial counts,
after the removal of biofilms, using an ultrasonic bath according to the procedures of the European
Biofilm Workgroup [21].

2.2 Direct staining of biofilms: Epifluorescence microscopy

The experimental work has addressed the observation of biofilms developed and attached on the surface
of glass beads. Different fluorochromes were evaluated to optimize the direct staining of biofilms for
fluorescence microscopy (Table 1). A Nikon Eclipse E-800 with 4 different filters was used for the ob-
servation of biofilm samples. To solve the problems due to the nature of the attachment support (glass
beads), a polycarbonate slide cell with a hole to contain and subject a 5 mm diameter glass bead was
designed.

2.3 Observation and biofilm analyses by confocal microscopy

Direct observations using a LEICA TCS-NT confocal scanning laser microscope (Leica Microsystems,
Deerfield, USA) were carried out to evaluate the bacterial colonization of the packed bed reactors. After
being stained using acridine orange (0.01%) or Live/Dead (Molecular Probes, Oregon) followed by 15
min. of incubation in the dark at room temperature, images of individual glass spheres stained with each
dye were obtained with the aid of the polycarbonate slide cell. The biofilms were viewed directly with oil
and coverslip.

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A series of several optical slices at different intervals were collected in each observation. These images
were then arranged serially and with the aid of the software provided with the LEICA TCS-NT confocal
laser microscope, all statistical height and surface parameters were calculated. The main surface parame-
ters determined for each sample were the maximum roughness depth (Rt), the arithmetical mean rough-
ness (Ra) and the root-mean square roughness (Rq). The thickness (height) of the biofilm developed on
glass beads is measured by means of consecutive optical thin sections in the xy axes. Finally, vertical
sections (xz position) of the biofilms developed on glass beads were also obtained.

Table 1 Different fluorochromes used for direct staining of biofilms.

Maximum
Stain Concentration Method References
Excitation/emission
Acridine Total cell
0,01% 503 /530-640 nm [22]
orange number
Viable Cell
0,05% CTC 365/602 nm number [16]
CTC/Dapi
1 µg/mL DAPI 347/ 456 nm Total cell [13]
number
Viable Cell
6 µM Syto 9
480/500 nm number [23]
Live/Dead 30 µM propidium
490/635 nm Total cell
iodide
number

2.4 Cryoembedding and cryosectioning of biofilms

We performed this technique, suitable for thick biofilms, using a simultaneous stain with DAPI and CTC
[13, 14, 16]. The samples were stained with 0.05% CTC for 2 hours at room temperature in the dark, and
with a slight agitation. After a fixation with 5% formaldehyde for 5 min., the glass beads placed on a dry
ice block were embedded with Tissue-Teck inclusion medium (Miles Incorporated 4583, Elkhart, USA).
When the inclusion agent became completely white –an indication of its freezing-, the embedding me-
dium was separated from the sample. After this, more inclusion agent was added at the exposed part of
the biofilm, and the upper part of the sample was marked. The biofilms embedded in the inclusion agent
were cut in a 5 µm thickness sections with a Leica CM 1800 cryotome (Leica Microsystems, Deerfield,
USA), mounted in a surface treated glass slide (Superfrost-Plus Microscope Slides, Fisherbrand 12-550-
15, USA). Finally, DAPI was added at the usual concentration (1 µg/mL) for 3 min, and samples were
air-dried.

2.5 Observation of biofilms with ESEM

Biofilm samples were also examined using the environmental scanning electronic microscopy (ESEM).
ESEM has all the advantages of the SEM, but at the same time can acquire high resolution images with
pressures between 0.1-50 Torr, allowing the examination of hydrated biofilms [24, 25]. Biofilms were
examined directly, without previous treatment, with an Electroscan 2020 (Philips Electron Optics, Eind-
hoven, The Netherlands). The ESEM was operated at 20 kev with the secondary electron detector (ESD),
and the chamber environment was adjusted to 7ºC, with a saturate vapor pressure of 7.51 Torr, in order
to maintain the hydrated condition of each biofilm sample.

2.6 Observation of biofilms with Confocal Perfilometer (PLµ)

Biofilm samples were also examined using a confocal laser perfilometer based on the depth from focus
principle [26, 27]. Using a white source light and a slit pattern instead of pinhole like conventional con-

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96 Jordi Morató et al.: Microscopy techniques applied for monitoring the development of aquatic biofilms

focal microscopes, it is possible to obtain the biofilm surface topography with a spatial resolution less
than 0,5 µm.

2.7 Statistical analysis

A normality test was performed in order to select parametric or non-parametric statistical tests. The sig-
nificance of the difference between means was calculated by the Mann-Whitney U test or by T-Student
test (giving a P value and 95% confidence intervals for the difference in mean rank).

3. Results and Discussion

3.1 Observations and analyses of biofilms by confocal microscopy

Figure 1 shows the evolution of the process of colonization on glass beads, after 25, 65 and 92 days of
exposition to the groundwater source. The main surface parameters determined for each sample permit-
ted the monitoring of the microbial colonization of each glass beads (Table 2). All studied parameters
have showed a significant increase (P<0,001) during the entire time of exposition to groundwater. At the
first stages, the glass spheres were rapidly colonized and appeared to form a monolayer biofilm, which
was initially localized and with an average thickness of 4.5 µm (SE=0.25, n=125), with large areas un-
colonized. When the biofilm matured, between days 30 and 65, it was possible to differentiate some cell
clusters, and bacterial growth filled-in the previously uncolonized zones. After 65 days, thick layers of
biofilm, which contained large amounts of polymer, covered some areas. At this stage a low basal layer
of cells could be distinguished, which was 10 µm in thickness (SE=0,12, n=100), with stacks of biofilm
coming up from the surface. The cell density, surface parameters and thickness analyzed from biofilms
developed on glass beads increased for the first 90 days (p<0.001). Some sections presented high cell
density regions simultaneously with spaces without any cells, which could correspond to void channels,
needed to improve the transport of nutrients to the inferior portions of the biofilm. The absence of colo-
nization between these stacks enabled flow of water and movement of protozoa within channels [1]. The
Rt values obtained seem to indicate that the plateau-phase of development could be reached after 90
days. Surface coverage measured from total cell counts [28] increased from a 20% after 25 days to a
100% after 65 days. At this later stage, the structure and morphology of the biofilms resembled the typi-
cal of mature ones and most of the glass beads were covered with a thick mushroom-shaped biofilm.
Total cell counts with DAPI or acridine orange increased significantly (P<0,001) during the development
of the biofilm, from 1,2x107 Cells/cm2 at day 27 to 7,74x107 Cells/cm2 at day 65, reaching a maximum
value of 1,27x108 Cells/cm2 after 92 days. The numbers of viable cells (determined in CTC stained sam-
ples) paralleled the total cell counts, increasing significantly (P<0,001), but with values 0,5-0,9 logs
lower than those obtained with DAPI counts. The percentage of viable cells significantly increased from
29.4% at 27 days to 35,1 after 65 days. Afterwards, viability decreased to 25.5%. The samples were also
stained with Live/Dead BacLight. The values encountered were 1-2 log smaller for total bacterial counts
when compared with DAPI counts, and the same applied for viable cells with CTC.

3.2 Observations by cryoembedding, cryosectioning and fluorescence microscopy

This technique had to be adapted to the special characteristics of the support material, the glass beads,
which were difficult to work with due to the curvature of their surface. Some of the replicates performed
(20% aprox.) were not suitable for carrying out microscopic observations, due to the difficulty of sepa-
rating the glass beads from the embedding medium. On the other hand, the abundance of abiotic compo-
nents, such as corrosion products, carbonate deposits and other inorganic precipitates, hindered the ob-
taining of adequate samples. Nevertheless, this methodology was applied successfully to mature biofilms
in which the simultaneous staining of samples with DAPI and CTC allowed to perform total and viable

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Current Issues on Multidisciplinary Microscopy Research and Education 97

bacterial cell counts while, at the same time, measuring the biofilm thickness (Fig. 2, Left). The sections
obtained showed the importance of non-cellular elements in biofilms, demonstrating the high hydrated
condition of the biofilms analyzed.

Table 2 Evolution of different roughness parameters of biofilms developed on glass beads.

Time Thickness Surface
Ra (µm) Rq (µm) Rt (µm) n
(days) (µm) (mm2)
25 4,54 ± 0,33 1,66 ± 0,20 1,47 ± 0,26 14,61 ± 1,72 1199,8 77
65 6,60 ± 0,5 1,95 ± 0,15 1,72 ± 0,16 16,10 ± 1,04 1092,8 61
92 10,16 ± 1,01 2,63 ± 0,34 1,89 ± 0,26 22,02 ± 2,50 359,5 36
190 - 3,56 ± 0,54 4,35 ± 0,56 18,77 ± 1,99 1797,4 20

3.3 Observations by Environmental Scanning Electron Microscopy (ESEM)

ESEM is a good technique to appreciate the different details of the architecture of biofilms (Fig. 2a,
Right). In the experimental conditions used, and after 190 days of exposition to groundwater, the entire
surface of the glass beads was colonized by a mature biofilm in which bacterial aggregates interspersed
with inorganic and organic material that constituted the matrix of the biofilm and sometimes hindered the
identification of bacterial cells. ESEM constitutes a suitable technique to study the biofilm architecture
of fully hydrated biofilms.

3.4 Observations by Confocal Perfilometry

The images acquired (Fig. 2b and 2c, Right) showed the high quality and versatility of this system. The
specification of this equipment allowed the acquisition of wide field images (max. 1.4x1.05 mm). The
super-long work distances used facilitate and improve the observation of non-flat surfaces, such as the
glass beads used in our reactors. Moreover, the utilization of a white light source does not affect the
viability and activity of the biological materials.

4. Summary
Biofilms developed on a packed bed reactors exposed to continuous groundwater flow were successfully
monitored along time using different microscopic techniques. Confocal microscopy is the most versatile,
applicable and suitable technology for biofilm study. Biofilm thickness and surface-height parameters
could be determined and, at the same time, the combined analyses of different horizontal (xy) sections,
vertical sections (xz) and stereoimages permits the analyses of the architecture of biofilms, identifying
the precise situation of microbial cells, channel voids and EPS materials. The enhancement of images by
means of digital treatment with the completion of z-projections –with a maximum intensity algorithm– is
a useful technique to perform a qualitative description of the colonized surface. This algorithm improves
the contrast and facilitates the differentiation between the colonized and uncolonized regions of the mate-
rial.
In some cases, mature biofilms treated with fluorochromes are only partially stained due to the inability
of the reagent to penetrate the lower layers of the biofilm. This problem has only been observed when
biofilms have Rt values higher than 25 µm. In these cases, and whenever the biofilm thickness was high,
cryoembedding has been used with success.
Confocal perfilometer could be considered a perfect equipment for a non-invasive monitoring of the
microbial colonization of different surfaces, and specially, for glass beads. The wide field images ob-
tained combined with the super-long work distances of the objectives used and the utilization of a white
light source facilitates and improves the observation of non-flat surfaces without affecting the viability
and activity of microbial cells.

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98 Jordi Morató et al.: Microscopy techniques applied for monitoring the development of aquatic biofilms

Fig. 1 Direct observations of biofilms developed on glass beads after 25 (a), 65 (b) and 92 (c) days of exposition to
groundwater (Objective 100x1,4 Oil-PLAPO): (left) Vertical sections (x-z), three replicates; (right) Z projection of a
stack of images, using a maximum intensity algorithm.

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Current Issues on Multidisciplinary Microscopy Research and Education 99

Fig. 2 (Left) Cryoembedding and cryosectioning of biofilms developed on glass beads after 65 days of exposition to
groundwater. Staining with DAPI and CTC (Objective 100x): (a), Bright field, (b), DAPI, (c), CTC. (Right) Obser-
vations of biofilms with: a) ESEM, biofilms developed after 190 days of exposition to groundwater, b, c) Confocal
perfilometer, biofilms after 92 days of exposition to groundwater, SLWD 10x.

ESEM could be useful as a complementary tool to help in the characterization of the structure and archi-
tecture of biofilms. In fact, ESEM could reveal the exact topography of intact, live and fully hydrated
biofilms, with a higher magnification than the other microscopy techniques.
Today a vast range of tools and equipments exists to move forward in the knowledge of live biofilms.
The different microscopy techniques applied in this work complement each other and allow us to im-
prove our comprehension of the composition, structure and architecture of aquatic biofilms.

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