Insight into the Microbial Multicellular Lifestyle via

Flow-Cell Technology and Confocal Microscopy

Sunje Johanna Pamp,* Claus Sternberg, Tim Tolker-Nielsen

� AbstractBiofilms are agglomerates of microorganisms surrounded by a self-produced extracellu-lar matrix. During the last 10 years, there has been an increasing recognition of biofilmsas a highly significant topic in microbiology with relevance for a variety of areas in oursociety including the environment, industry, and human health. Accordingly a numberof biofilm model systems, molecular tools, microscopic techniques, and image analysisprograms have been employed for the study of biofilms under controlled and reproduc-ible conditions. Studies using confocal laser scanning microscopy (CLSM) of biofilmsformed in flow-chamber experimental systems by genetically color-coded bacteria haveprovided detailed knowledge about biofilm developmental processes, cell differentia-tions, spatial organization, and function of laboratory-grown biofilms, in some casesdown to the single cell level. In addition, the molecular mechanisms underlying theincreased tolerance that biofilm cells often display towards antibiotic treatment arebeginning to be unravelled. ' 2008 International Society for Advancement of Cytometry

� Key termsbiofilm; confocal microscopy; fluidic device; fluorescent reporter; subpopulation

BIOFILMS: THE MICROBIAL MULTICELLULAR LIFESTYLEMicroorganisms constitute the most successful form of life on earth, in terms of

total number, phylogenetic diversity and extent of habitats colonized. They impact

on human existence and well-being, either directly by influencing human develop-

ment, health and disease, or indirectly by carrying out processes in the natural envir-

onment or man-made environments (1–3). Our present state of knowledge on the

biology of microbial cells is, due to historical reasons, largely a result of empirical

research work on microbial cells living in suspension in a liquid growth medium.

However, it is now generally acknowledged that the majority of microbial cells on

earth are living in spatially distinct communities, referred to as biofilms. Biofilm cell

populations in many cases exhibit distinct features compared to planktonic cell

populations (1–5). Therefore, research work aimed at unraveling characteristics of

the microbial multicellular lifestyle will provide a more complete understanding and

definition of microbial life, and ultimately also human life, in nature.

Microbial Multicellular Communities

Biofilms come in a great variety of sizes and shapes. Some of the most common

types contain mushroom-like, pillar-like, hilly, or flat multicellular structures. These

structures are formed by cells that are held together by interconnecting compounds,

such as self-produced polysaccharides, proteins, extracellular DNA, and cell lysis pro-

ducts, as well as matter from the immediate surrounding environment, which alto-

gether constitute the so-called ‘‘matrix.’’ This allows cells to form long-term relation-

ships, interact with each other and establish metabolic cooperations (6,7). Biofilms

in nature are generally beneficial and frequently established on hydrous solid and

semi-solid surfaces, such as soil, rock material, or surfaces of animals and plants. Bac-

Department of Systems Biology,Technical University of Denmark, Lyngby,Denmark

Received 19 August 2008; Accepted3 November 2008

Grant sponsor: Danish research councils.

*Correspondence to: S€unje JohannaPamp, Department of Microbiology andImmunology, Stanford University Schoolof Medicine, VAPAHCS, 154T, Building101, B4-185, 3801 Miranda Avenue, PaloAlto, CA 94304, USA.

Email: pamp@stanford.edu

Published online 2 December 2008 inWiley InterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20685

© 2008 International Society forAdvancement of Cytometry

Review Article

Cytometry Part A � 75A: 90�103, 2009

terial communities play key roles in food webs in nature.

Many of the underlying processes are interdependent and

require cooperation between various bacterial species with dif-

ferent metabolic capacities (3,4,6,8,9). The fact that in biofilms

the participating microbial members are situated in close

proximity seems to be advantageous, since metabolites can

easily be transferred and metabolized further. In cases of

adverse conditions such as desiccation, osmotic shock, or ex-

posure to toxic compounds, UV radiation, or predators, the

microbial community as whole can provide protection. More-

over, multicellular communities provide ideal conditions for

horizontal gene transfer, which is important for microbial evo-

lution and genetic diversity (4,6,10,11). However, in many

man-made environments, such as industrial or medical set-

tings, the formation of sessile microbial communities, for

example, in production lines or on indwelling medical devices

is unwanted and detrimental. Resultant energy losses, corro-

sion, fouling, persistent infections, and potential death of

humans cause an enormous socioeconomic burden each year

worldwide (6,12).

Microbial Communities as Inhabitants of Humans:

Impact on Health and Disease

Microbial communities natively populate human mucous

membranes and epithelial surfaces like the gastrointestinal

tract, oral cavity, and skin. Each of the body sides is colonized

with a mixed microbial community of characteristic composi-

tion (2,13,14). Intriguingly, for most of our lifespan we do not

suffer from harboring these microbial communities. In fact,

they are important and beneficial to us as they can degrade

nutrients and thereby making them accessible to us, and

synthesize some vitamins, which we have not evolved to

synthesize on our own. Moreover, these communities play key

roles in the development of our immune system and anatomy

of the mucosal surfaces and exert protective functions against

exogenous pathogens (2,15,16).

The relationship between the host and its microbial com-

munities is delicately balanced but under certain conditions, it

can break down and result in infectious diseases. These infec-

tions can be caused either by members of the indigenous

human microbial community or by microorganisms from the

environment (1,2,6). Under conditions where the host is

impaired, for example immunocompromized, injured, or suf-

fering from cancer or cystic fibrosis, harmful biofilms can de-

velop at different body sides and cause persistent infections.

Among those infections are various device-related infections,

pulmonary infections, periodontitis, wound infections, otitis

media, osteomyelitis, infective endocarditis, and chronic pros-

tatitis (5,17–19). Bacteria, which have been found to be

involved in human biofilm-related infections, are for example

Pseudomonas aeruginosa, Staphylococcus spp., Escherichia coli,

Salmonella spp., Enterococcus spp., Streptococcus spp., Proteus

mirabilis, Klebsiella spp., Enterobacter spp., and Haemophilus

influenza. Biofilm-related infections are in many cases persis-

tent, that is, they evidently cannot be eradicated by the host

immune system and they are difficult to eradicate by antimi-

crobial chemotherapy (5,17–20).

Cultivation and Analysis of Multicellular Communities

Under Laboratory Conditions

Biofilms are intriguing societies of microbes and it is of

general interest to unravel the processes involved in their de-

velopment, physiology, and adaptation to perturbations. Em-

pirical research work on biofilm biology will ultimately reveal

new strategies that contribute to maintenance and restoration

of human health through well-informed manipulations of mi-

crobial communities. However, due to their complexity, nat-

ural microbial communities have been challenging objects of

investigation. In addition, biofilms are often located at places

that are difficult to access, which makes direct and continuous

examinations difficult. Various factors impact on biofilm biol-

ogy and many of those factors are interrelated. To reduce com-

plexity and facilitate investigations in the laboratory under

controlled and reproducible conditions, a number of ‘‘simple’’

biofilm model systems have been established. These include

flow-cell-grown biofilms, colony biofilms, microtiter dish-

grown biofilms, and pellicle biofilms (21–23). To discover

genetic determinants and regulatory pathways impacting on

the biofilm mode of life, major focus relies on well-character-

ized and genetically tractable microorganisms, such as Pseudo-

monas aeruginosa. The gold standard in biofilm research is an

approach, which involves flow-cell technology in combination

with confocal laser scanning microscopy (CLSM). Unlike

other techniques, this particular methodology allows getting

insight into details of developmental processes, spatial organi-

zation and function of laboratory-grown biofilms in real-time

under continuous and non-invasive conditions down to the

single-cell level (21,24,25).

CULTIVATION OF BIOFILM CELLS IN A FLUIDIC DEVICE

Biofilm flow-cell setups allow the cultivation of biofilms

under continuous hydrodynamic conditions. The biofilm

flow-cell system consists of five major components: A medium

reservoir, a multichannel peristaltic pump, bubble traps, flow-

cells, and an effluent reservoir. All parts are consecutively con-

nected via silicone tubings, splitters and connectors (Fig. 1A)

(22).

The Biofilm Flow-Cell

A central component of the biofilm flow-cell system is

the flow-cell, providing chambers for biofilm cultivation. A

number of different flow-cell designs exist. Widely used is the

flow-cell described here, which is a modified version of a flow-

cell originally developed by Wolfaardt et al. (26). The flow-cell

is designed so that it can be mounted on nearly any optical

microscope. It consists of two parts, a flow-cell base (Fig. 1B)

and a conventional microscopy glass coverslip. The flow-cell

base is made of a polycarbonate part in which parallel chan-

nels with individual dimensions of 40 3 4 3 1 mm have been

drilled. In the flow-cell presented here three individual chan-

nels have been drilled, meaning that in one flow cell three

individual (parallel) biofilm experiments can be performed.

To both ends of each channel, ports of 1 mm diameter have

been drilled, which serve as medium inlet and effluent outlet,

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Cytometry Part A � 75A: 90�103, 2009 91

respectively (22). A microscopy glass coverslip (50 3 24 mm)

is placed on top of the flow-cell base and thereby covering the

open side of the channels to form closed channels. The cover-

slip is glued with silicone on the flow-cell base by applying sili-

cone glue as thin strings on top of the base along the perimeter

of the channels. The microscopy coverslip basically fulfills

three functions: it serves as one wall of the channels thereby

forming a closed flow channel, it serves as substratum for bio-

film formation, and it is optically compatible with micro-

scopic examination techniques (21,22,24,27).

The Biofilm Flow-Cell System

To eventually continuously supply the biofilm cells with

nutrients, the flow-cell needs to be connected with the remain-

ing components of the flow-cell system (Fig. 1A). Before the

medium reservoir is connected, possible contaminants are

removed from the entire system. This is achieved by disinfec-

tion of the system with sodium hypochloride or ethanol, or by

sterilization with ethylene gas. Most components are also com-

patible with autoclaving at 1218C. Subsequently the reservoir

with sterile nutrient medium is connected to the system. The

composition of the medium is chosen dependent on the

requirements of the organism(s) of interest. The system is

filled with medium using a peristaltic pump. Conventional

peristaltic pumps do not deliver an entirely pulseless flow, in

contrast to multiroller peristaltic pumps. If the carbon source

in the medium has a high hydrophobicity then medium is run

through the system for an extended period to equilibriate the

silicone tubings, before introducing the organisms of interest

(21,22,24,27).

The organisms of interest are introduced at desired initial

optical density into the flow chambers via a syringe while me-

dium flow is paused. The syringe is inserted upstream of the

flow-cell through the silicon tubing into the inlet of the flow

chamber, the cell suspension of microorganisms is injected

and the resulting fine hole in the silicone tubing is sealed with

a drop of silicone glue. Microbial cells are allowed to attach to

the substratum for 1 h, whereupon medium flow is resumed

and adjusted to laminar flow. For the biofilm system described

here, the flow rate is often set to 0.2 mm/s (approximately 3

ml/h/channel) (21,22,24,27). Universal equations for the cal-

culation of hydrodynamic parameters in a flow-cell can be

found elsewhere in the literature (e.g., 28). While fresh me-

dium is continuously transported into the flow cells to allow

biofilm development and differentiation, effluent is trans-

ported out and collected in the reservoir placed at the most

downstream part of the system.

Under certain circumstances air bubbles might arise in

the system, which can remove biofilm cells from the substra-

tum while passing through a flow channel. To prevent this,

bubble traps have been designed and are mounted between

the peristaltic pump and the flow-cells. Bubble traps are com-

posed of three parts: a bubble trap base made of a polycarbo-

nate part (Fig. 1C), a syringe cylinder, and a lid to close the

top of the syringe cylinder. Any air bubbles that might pass

into the bubble trap will float to the top of the syringe cylin-

der, and are prevented from passing through the downstream

part of the system. Note that the inlet of the bubble trap base

is designed so that it is situated higher than the outlet part

(Fig. 1C).

The biofilm setup is compatible with various visualiza-

tion and quantification techniques (see following text). Cell–

cell interactions in a biofilm can be studied between (i) cells of

a single strain (e.g., 29), (ii) cells of a number of different

Figure 1. The biofilm flow-cell system allows the cultivation and

analysis of biofilm cells under continuous hydrodynamic condi-

tions. The system consists of five major components: a medium

reservoir, amultichannel peristaltic pump, bubble traps, flow-cells,

and an effluent reservoir. All parts are consecutively connected via

silicone tubings, splitters and connectors (A). Working drawing of

the flow cell base (design copyright DTU-Biosys). Reproduced

from Curr Protoc Microbiol 1B.2.1—1B.2.15 with permission fromJohnWiley& Sons, Inc. (B).Working drawing of the advanced bub-

ble trap base (design copyright DTU-Biosys). Reproduced from

Curr Protoc Microbiol 1B.2.1—1B.2.15 with permission from John

Wiley & Sons, Inc. (C). [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

REVIEW ARTICLE

92 Insight Into Biofilms via Confocal Microscopy

strains (e.g., mutants) of a particular single species (e.g.,

30,31), (iii) cells belonging to different bacterial species (e.g.,

32–35), and (iv) cells belonging to different domains, such as

bacterial cells and eukaryotic cells (e.g., 36). Activities in nat-

ural multicellular microbial consortia might also be studied in

flow-cells after harvesting samples from the natural environ-

ment, and transplanting the microbial cells into flow-cham-

bers where they are subjected to further analysis (e.g., 26,37).

The setup might be modified, for example, to study the effect

of substratum coatings on biofilm development and physiol-

ogy, or to study the effect of oxygen up- or down-shifts on

biofilm cells (38–40). Biofilm cells might also be harvested

from the flow chambers and subjected to further analysis, for

example global transcriptional analysis, fluorescence activated

cell sorting (FACS), or c.f.u. determinations (31,41,42). The

entire biofilm flow-cell system can be placed on a rolling table

to facilitate secure transport of the system for example

between an incubator room with a constant temperature (of-

ten 308C or 378C) and the room in which microscopic exami-

nations of the biofilms are taking place.

ANALYSIS OF BIOFILM CELLS

Because biofilms are complex 3-dimensional structures

the analysis of them is not trivial. While microbial single cells

easily can be monitored using a conventional microscope, bio-

films require additional resolution in the direction vertical to

the substratum (the z-axis). Here we describe the use of confo-

cal laser scanning microscopy (CLSM), fluorescent labeling of

biofilm cells, and image analysis in biofilm research.

Confocal Laser Scanning Microscopy (CLSM)

The use of the confocal laser scanning microscope has

helped overcoming the apparent shortcomings of the conven-

tional light microscope (e.g., the presence of out-of-focus

light) by introducing point illumination and a pinhole, which

allows optical sectioning of the specimen. The individual opti-

cal sections are subsequently assembled by aid of advanced

computer software (some examples are listed in the following

text) to a virtual 3D image (e.g., Figs. 2A and 2B and Fig. 3).

Typically a biofilm with a thickness of more than �150 lmcannot be rendered with a reasonable detail due to physical

factors. The implementation of multiphoton excitation is a

major step forward. Using a pulsed laser it is possible to guide

two (or more) photons to excite a fluorophore simultaneously.

This means that the energy of the photons is combined to

excite the target molecule. Using this technique the depth re-

solution (i.e., the minimum distance to resolve two points) is

many fold increased, from around 500–700 nm for standard

confocal microscopy to 100 nm for two-photon microscopy

(43). Neu and co-workers demonstrated two-photon confocal

imaging of thick z-sections with very high resolution (43,44).

Recently a new technology, stimulated emission depletion

(STED) has been developed into commercial products,

increasing the optical resolution even further by using two-

photon excitation in combination with quenching of near-by

fluorescence which otherwise could deteriorate the image

(45,46). STED, however, is currently of limited use since

only few fluorophores are suitable for this particular laser

excitation.

Fluorescent Labeling of Biofilm Cells

Confocal microscopy and derived methods require the

specimen to be fluorescent. The biofilm must therefore either

be autofluorescent by means of indigenous fluorescent mole-

cules, or the biofilm cells must express a fluorescent protein

(e.g., the green fluorescent protein, Gfp (47)), or individual

biofilm cells or other components of the multicellular struc-

ture must be stained. Early biofilm studies by the Caldwell

group employed a simple, yet efficient way of detecting the

biomass in flow cells: The void volume, that is, the liquid

phase was supplemented with a solution of fluorescein iso-

thiocyanate (FITC), leaving the biomass unstained. The result-

ing images were ‘‘negatives’’ and the biofilm could be rendered

as the dark portions of the images (48,49). This gave suffi-

ciently high resolution to determine for example cell sizes and

spatial relations. More recently developed stains, such as the

Syto stains (Invitrogen, Carlsbad, CA), can efficiently stain

cells in virtually any color of the rainbow. In combination with

propidium iodide (PI) it is further possible to specifically stain

live and dead cells. The dye Syto 9 will stain all cells green

regardless if they are dead or alive, while it is generally

assumed that only cells with a damaged membrane will be

stained by the red PI dye, indicating dead cells. Recent results

suggest that propidium iodide might be of limited use as cell

viability indicator for some environmental bacterial species

(50). Therefore, for each individual bacterial species a fine-

tuning of the dye combinations is recommended prior to per-

forming the actual experiments using mixtures of live and

dead cells of known ratios (51). Recently the assumption that

propidium iodide only targets dead cells was confirmed also

for biofilm cells that had been exposed to a membrane dama-

ging agent, using cell sorting of harvested biofilm cells and

re-growth test of the separated green (Gfp-tagged) and red

(PI-stained) cells on nutrient agar. Only the cells that were not

labeled red with PI were able to grow (42, and unpublished

results). Stains targeting the extracellular matrix such as lectins

(52,53) or calcofluor white (54,55) can also be employed to

visualize the surrounding of the biofilm cells. In addition, the

extracellular DNA component of the matrix can be visualized by

the use of different DNA-binding fluorophores (e.g., Ref. 56).

If genetic manipulation of the biofilm cells is possible,

chromosomal tagging of cells with a gene cassette encoding

the green fluorescent protein (Gfp) can be a useful option

(e.g., 57). Alternatively, plasmids encoding for the Gfp might

be introduced into the cells prior to biofilm examinations.

Depending on the construction, this fluorescent tagging can

be used as a simple labeling to verify the location of the cells

in a biofilm, or, by selecting suitable variants of gfp genes and

promoters, it can be used for monitoring gene expression in

biofilms. Such tagging of biofilm cells has been done to moni-

tor metabolic/physiological activity in biofilms by introducing

constructs encoding for Gfp derivatives with a short half-life,

placed under transcriptional control of a ribosomal promoter

(58). For example, the gfp[AGA] gene, encoding for a green

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Cytometry Part A � 75A: 90�103, 2009 93

fluorescent protein (Gfp) with a short half life, was placed

under transcriptional control of the ribosomal promoter

rrnBP1 and introduced into either E. coli or Pseudomonas spp.

wild type strains. Cells which have a high metabolic/physio-

logical activity can be expected to exhibit high Gfp[AGA]

expression and emit a high fluorescent signal, whereas cells

which have a low metabolic/physiological activity can be

expected to exhibit a low or no expression of the fluorescent

protein (41,58). Further, using Gfp variants with different

emission spectra, such as the Cfp (cyan fluorescent protein),

Yfp (yellow fluorescent protein), and Rfp (red fluorescent

protein), the spatial distribution of either cells in a multi spe-

cies biofilm can be determined, or of a number of (mutant)

strains of a single species tagged with different colors

(30,31,59,60).

Another way of fluorescently labeling biofilm cells is

through the use of fluorescent in situ hybridization (FISH),

where specific probes hybridize to the 16S rRNA in the cells.

For FISH a DNA probe is designed to match a distinct region

of the cell’s ribosomal RNA. The probes can be conjugated to

a fluorescent dye, such as fluorescein isothiocyanate (FITC) or

Rhodamine, or to an enzyme (e.g., horseradish peroxidase),

which deposits fluorescent molecules. It might be challenging

to introduce larger conjugates such as the horseradish peroxi-

dase into cells of thicker biofilms without destroying biofilm

cells due to harsh permeabilization procedures. Therefore

FISH involving probes with larger conjugates might preferen-

tially be applied on thin sections of thick biofilms. The num-

ber of ribosomes present in a given cell is proportional to the

growth potential of the cell, and FISH labeling can conse-

quently also be used to determine the growth status of a bio-

film cell (61). However, under certain conditions, for example,

stress, cells might have increased numbers of ribosomes,

although their actual growth rate is low. The probe design can

be adjusted so that the probe only labels a single species by tar-

geting a so called variable rRNA region, or a probe can label

all cells belonging to the same domain or phylum by choosing

a more conserved region. An example of such a probe is the

widely used EUB338, which can hybridize to virtually all bac-

teria (62). However, the growing knowledge on ribosomal

RNA encoding sequences has revealed that probes formerly

believed to be universal fail to be able to hybridize to species

or entire phyla of microorganisms in the realm they originally

were thought to cover completely (63).

Image Analysis

The recorded microscopic images can be used immedi-

ately or processed further for presentation or quantitative

analysis. The images that originate from a confocal micro-

scope are usually grayscale bitmap images, one from each focal

plane and one for each detection channel (color). An image of

a 30-lm-thick biofilm recorded with a step size of 0.5 lm in

three channels will result in 30/0.5 3 3 5 180 individual

images. Most microscope software pack the images in contain-

ers such as the LIF file format for Leica and the LSM format

for Zeiss microscopes. Furthermore the images will be rela-

tively large. In the example above the standard resolution of

256 gray tones and 512 3 512 pixels will result in a file with

the size of 45 MB. Special software is required to handle these

files and to render the beautiful biofilm representations. While

several packages are available, a few seem to dominate the

Figure 2. Analysis of a young mixed color-coded biofilm via con-

focal laser scanning microscopy (CLSM). Top—down view of ini-

tial multicellular structures (16 h old) formed by P. aeruginosawild type (Gfp-tagged), P. aeruginosa pilA (Cfp-tagged), and P.aeruginosa rhlA (Yfp-tagged). A special detector system mounted

on the CLSM allowed the simultaneous detection of the three clo-

sely related spectral variants of the green fluorescent protein, as

described in (31) (A). Separate display of initial multicellular struc-

tures by each individual tagged P. aeruginosa strain of the mixedcolor-coded biofilm (B). The top—down view images in (A) and

(B) are generated using a z-stack, i.e., here a series of single opti-

cal section image scans (n 5 15) in the xyz dimension, acquired ofthe initial multicellular structures (C).

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94 Insight Into Biofilms via Confocal Microscopy

market: Imaris (Bitplane, Bern, Switzerland), Amira (Visage

Imaging, Carlsbad, CA), Volocity (Improvision, Coventry, the

UK), Voxblast (Vaytek, Fairfield, IA), and Metamorph (Molec-

ular Devices, Sunnyvale, CA) (see 43 for a review). Designed

for the visualization of larger eukaryotic cells, most 3D presen-

tation packages are not optimized for the small cell sizes

involved in microbial biofilms. The average size for a Gram-

negative bacterium is 1 lm by 2–3 lm. The optical resolution

of a confocal image recorded with one-photon excitation is at

best 0.48 lm (64). This means that a sampling of images with

a smaller step size than this will not provide more informa-

tion. Consequently, a single bacterium will not appear in more

than one or two independent focal plane images, making a 3D

reconstruction of the single cell difficult. However, the biofilm

as a whole is much larger and can be rendered in 3D, although

it may not be possible to locate the individual cells. Typically

a step size of 1 lm is used for bacterial biofilms (e.g., Ref. 30).

The visualization software packages also include tools for

cleaning up the recordings, such as filters for noise and cross-

talk (the situation where one fluorophore is recorded in the

detection channel of another fluorophore). The main features

of these softwares are, however, their capability to visualize

the spatial organization of the recorded data. The rendering

can be in perspective 3D, or as 2D images in all three axes,

x-y, x-z and y-z. Sequential recordings over time can also be

rendered, and animated, providing a four-dimensional data-

set, x-y-z-t. Such data sets can quickly be very large (some-

times several gigabytes) and it sets new requirements for the

software and hardware. Special detector systems mounted on

the confocal microscope might also facilitate the simultane-

ous separation of a number of fluorescent spectra (lambda

mode configurations) originating from different fluorescent

molecules or proteins with overlapping emission spectra,

increasing the possibilities for multi fluorescent labeling of

biofilms (Fig. 2) (31).

Quantitative analysis of 3D images can be challenging

and several groups have developed special software packages

for this purpose. The algorithms start by determining the

extent of the biomass, by thresholding each focal plane

image. This step is crucial and much effort has been put into

optimization of it. It can be done either manually, semi- or

fully automated. Some of the first attempts to provide robust

quantification software were done by Yang et al. (65) and

Heydorn et al. (66) with the programs ISA and COMSTAT,

respectively. Both extract a number of parameters, which can

be used to characterize the biofilm: biomass (pixels occupied

by biomass), biofilm height, height distribution, roughness

coefficient, and diffusion distances, to mention a few. Both

programs are developed in MATLAB (MathWorks, Natik,

MA). COMSTAT utilizes a command-line interface within

the MATLAB shell, which is required for operation, whereas

ISA and its successor ISA3D are compiled programs that do

not require the MATLAB package. Other quantification soft-

ware is available, for example, the web-based PHILIP (67), a

program that has a higher level of automation than the ISA

and COMSTAT packages. Further developments of PHILIP

have taken the automation and robustness towards the

threshold function to a new level (68,69). COMSTAT is now

available in a complete reprogrammed version 2, which is

running on the platform independent software foundation

Java (unpublished results). This new version of COMSTAT

software uses the same thresholding algorithms as the PHI-

LIP software and also incorporates a number of new features

such as a user programmable plug-in interface for end user

Figure 3. Development of multicellular structures by P. aerugi-nosa. Initial multicellular structure (1.5 days old): a microcolonyformed by P. aeruginosa pilA cells (Cfp-tagged) surrounded by

motile P. aeruginosa wild type cells (Yfp-tagged), which are be-ginning to colonize the microcolony. Grid size: 5 lm (A). Mature

multicellular structure (4 days old): mushroom-shaped multicellu-

lar structures with P. aeruginosa pilA cells (Cfp-tagged) formingthe stalk, and P. aeruginosa wild type cells (Yfp-tagged) formingthe cap. Grid size: 20 lm (B). The confocal laser scanning micro-

graphs show a 3D image view of the multicellular structures,

respectively.

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Cytometry Part A � 75A: 90�103, 2009 95

defined image processing functions, and a wider range of

image formats.

DEVELOPMENT OF MULTICELLULAR STRUCTURES IN

PSEUDOMONAS AERUGINOSA BIOFILMS

Studies involving flow-chamber technology and CLSM

have provided knowledge about biofilm formation of numer-

ous bacterial species of both environmental and medical rele-

vance. The studies have, among many other things, given

information about the mechanisms, environmental cues and

underlying genetic elements involved in attachment of cells to

surfaces, formation of multicellular structures, and dispersal

of cells from multicellular structures. In the present section we

present examples of work done on these topics with biofilms

formed by the opportunistic pathogen P. aeruginosa.

Transport of Cells to the Surface

The bacterial cells can reach a surface prior to coloniza-

tion by means of passive motility mediated by brownian

motion or vortex currents, or by active swimming motility

mediated by flagella rotation (70). Flagella-driven motility has

been reported to enhance the efficiency of surface colonization

by P. aeruginosa (71,72). The dependence on flagella is condi-

tional however, as non-flagellated P. aeruginosa mutants and

the isogenic wild type were shown to attach equally well to a

surface in flow-chambers under some conditions (29).

Attachment of Cells to the Surface

P. aeruginosa appears to possess a number of different

adhesins that can function in attachment to a surface. The

conditional dependence on flagella for surface colonization

described above may, besides a role in bacterial transport to

the surface, be because of adhesion properties of the flagella. P.

aeruginosa cells were shown to attach apically to the glass sur-

face in flow-chambers and rotate, indicating that the initial

attachment occurred by means of flagella (72). The initial

attachment was reversible, but cells became irreversibly

attached by progressing from apical to longitudinal attach-

ment. The gene sadB (PA5346) was shown to be required for

the progression from apical to longitudinal attachment (73).

Non-flagellated mutants of P. aeruginosa attached longitudin-

ally (72), indicating that flagella are not required for attach-

ment per se but may enhance the process. Type IV pili were

shown to be important for attachment of P. aeruginosa to vari-

ous surfaces (74–76), and hyperpiliated variants of P. aerugi-

nosa were shown to rapidly initiate formation of strongly

adherent biofilms (75). The dependence on type IV pili for

cell attachment appears to be conditional, however, as non-

piliated P. aeruginosa mutants and the isogenic wild type were

shown to attach equally well to surfaces in flow-chambers

under some conditions (29). In addition to flagella and type

IV pili, fimbrial appendages termed Cup fimbria were shown

to play a role in surface-attachment of P. aeruginosa (77), and

evidence has been provided that extracellular DNA plays a role

in surface attachment of P. aeruginosa cells under some condi-

tions (78).

Formation of Initial Multicellular Structures

Shortly after attachment to the surface in flow-chambers

the P. aeruginosa cell population consist of a non-motile sub-

population, and a motile subpopulation that moves on the

surface via type IV pili or flagella activity (29,30,79,80).

Experiments with mixtures of Cfp-tagged and Yfp-tagged cells

provided evidence that microcolonies develop by clonal

growth in the flow-chambers (30). The initial microcolonies

are formed by cells, which do not display motility and there-

fore proliferate at fixed positions, while the cells that move on

the surface do not participate in formation of the initial

microcolonies (29,30). Conditions that promote extensive

motility of P. aeruginosa have been shown to prevent micro-

colony formation in flow-chambers. Interestingly, this could

be a mechanism which prevents biofilm formation in humans

since lactoferrin, a component of the innate immune system,

was shown to induce extensive type IV-driven motility in P.

aeruginosa (79).

The cellular adhesiveness mediated by the factors

involved in surface attachment probably also plays a role as

cell-to-cell adhesin in the earliest phase of microcolony forma-

tion. For example, the small microcolonies formed by P. aeru-

ginosa in the initial phase of biofilm formation could be dis-

persed by exogenous DNase activity indicating a role for extra-

cellular DNA as matrix component in the early phase of P.

aeruginosa biofilm formation (78).

Formation of Mature Multicellular Structures

Although the relative importance of the mechanisms

involved in initial formation of multicellular structures

appears to vary dependent on the conditions, the outcome of

initial biofilm formation seems to be similar under various

conditions. The early P. aeruginosa biofilm generally consists

of a subpopulation of non-motile cells forming small microco-

lonies, and a subpopulation of motile cells, which move on

the surface between the small microcolonies. After formation

of the initial microcolonies structural biofilm development by

P. aeruginosa depends on the prevailing conditions. For exam-

ple, flat biofilms are formed in flow-chambers irrigated with

citrate minimal medium (29), while heterogeneous biofilms

containing mushroom-shaped multicellular structures are

formed in flow-chambers irrigated with glucose minimal

medium (30).

Formation of the flat P. aeruginosa biofilm in flow-cham-

bers irrigated with citrate medium was shown to occur via ex-

pansive surface-migration of cells from the initial microcolo-

nies (29). CLSM time-lapse microscopy indicated that a shift

from non-motile to migrating cells occurred when the initial

microcolonies reached a certain size, suggesting that the shift

may by induced by a limitation arising in the initial microco-

lonies (For representative movies the reader is referred to Ref.

81). Migration of the bacteria appeared to cease with matura-

tion of the citrate-grown biofilm. Since biofilm formation by a

P. aeruginosa pilA mutant (deficient in biogenesis of type IV

pili) occurred without the expansive phase, and resulted in

discrete protruding microcolonies, it was suggested that the

REVIEW ARTICLE

96 Insight Into Biofilms via Confocal Microscopy

expansive migration of the cells on the surface was type IV

pili-driven (29).

Formation of the mushroom-shaped structures in glu-

cose-grown P. aeruginosa biofilms evidently occurs in a se-

quential process where the initial microcolonies formed by the

non-motile subpopulation become colonized by cells from the

migrating subpopulation that subsequently form mushroom

cap-like structures on top of the initial microcolonies, which

then become mushroom stalks (for representative movies the

reader is referred to Ref. 82) (30,42). Growth of the initial

microcolonies in the glucose-grown biofilms continue past the

point where spreading by type IV-driven motility prevents fur-

ther microcolony formation in the citrate-grown biofilms. In

glucose-grown biofilms containing a mixture of Cfp-tagged

and Yfp-tagged P. aeruginosa wild type cells, mushroom-

shaped structures are formed that have single-color stalks and

two-color caps, in concordance with the stalks being formed

by proliferation of non-motile cells, and the caps being formed

via aggregation of motile cells (30). In biofilms containing a

mixture of P. aeruginosa wild type and P. aeruginosa pilA mu-

tant, the pilA mutants can only form stalks whereas the wild-

type cells form the caps, suggesting that type IV pili are neces-

sary for cap-formation (30). Figure 3A shows a microcolony

formed by Cfp-tagged pilA mutants surrounded by Yfp-tagged

motile wild type cells, which are beginning to colonize the

microcolony. Figure 3B shows mature mushroom-shaped

structures with Cfp-tagged pilA mutants in the stalk and Yfp-

tagged wild type cells in the cap. In addition to type IV pili,

formation of the mushroom cap evidently also depends on fla-

gella, as P. aeruginosa fliM mutants (deficient in biogenesis of

flagella) were shown to be deficient in cap-formation (83).

Evidence was provided that flagellum-driven surface-asso-

ciated motility is involved in cap-formation, whereas the de-

pendence of cap-formation on type IV pili may be due to

binding of these pili to extracellular DNA which is particularly

abundant on the microcolonies that become colonized during

mushroom-structure formation (as described in the following

text). In accordance with a role of type IV pili-driven motility

in the early phase of biofilm formation and a role of flagella-

driven motility in the later phase of biofilm formation, fla-

gella-driven surface associated motility (swarming) is known

to depend on quorum sensing (e.g., 84), that is, a mechanism

by which bacteria can monitor their cell population density

through the extracellular accumulation of signaling molecules

and express genes when the cell density is high. Production of

biosurfactants, which is under quorum sensing control, was

shown to facilitate formation of the mushroom cap structures

(31). Migration of the bacteria appears to cease with matura-

tion of the glucose-grown biofilm.

Experiments involving the fluorescent reporter rrnBP1-

gfp[AGA] (see preceding text) provided evidence that meta-

bolic/physiological activity is highest in cells forming the cap-

subpopulation, and lowest in cells forming the stalk-subpopu-

lation (41). As can be seen in Figure 4A, the cells of the cap-

forming subpopulation exhibit a high fluorescent signal, indi-

cating high metabolic activity, whereas cells of the stalk-form-

ing subpopulation exhibit a low fluorescent signal, indicating

low metabolic activity (41). In the control biofilm formed by a

P. aeruginosa wild type strain expressing the stable version of

Gfp, all cells exhibit similar levels of fluorescence emission

(Fig. 4B). This spatial distribution of high and low metaboli-

cally active cells seems plausible as cells in the cap part can

obtain oxygen and nutrients from the bulk liquid to drive

metabolic processes such as replication, transcription and

translation, in contrast to the cells in the stalk part where con-

centrations of dissolved oxygen and nutrients are likely to be

low. It might be speculated that higher levels of oxygen and

nutrients in the bulk liquid are sensed by some cells during

biofilm development, and that this results in an attraction,

driving cells to migrate on top of microcolonies formed by

non-motile cells. In support of this, P. aeruginosa strains de-

fective in chemosensory systems show defects in cap-forma-

tion [(83) and S.J.P and T.T.N., unpublished].

The multicellular structures in P. aeruginosa biofilms are

stabilized by a matrix consisting of exopolysaccharides, extra-

cellular DNA, and proteins. The alg operon (PA3540-PA3551)

encoding alginate polysaccharide appears not to be expressed

in P. aeruginosa flow-chamber biofilms (85), but evidently

plays a role in biofilm formation by P. aeruginosa in particular

in the lungs of cystic fibrosis patients (86). The psl genes

(PA2231-PA2245) encode production of a mannose rich exo-

polysaccharide, which was shown to play a role in P. aerugi-

nosa biofilm formation (87–89). The pel (PA3058–PA3064)

genes encode production of a glucose rich matrix component,

which was shown to facilitate biofilm formation of some P.

aeruginosa strains (90). Extracellular DNA was shown to be

present in high concentrations particularly on the microcolo-

nies in young P. aeruginosa biofilms and between the stalk-

forming and cap-forming subpopulations in mature biofilms

(56). Type IV pili bind to DNA (91,92), and evidence has been

presented that the high concentration of extracellular DNA on

the mushroom stalks may cause accumulation of the migrat-

ing piliated cells and thereby facilitate formation of the mush-

room caps (83). Production of large amounts of extracellular

DNA in P. aeruginosa biofilms has been shown to be depend-

ent on the PQS quorum-sensing system (56). Evidence is

accruing that Cup fimbria in addition to their role in initial

biofilm formation also play a role as matrix components in

mature biofilms. The sadARS genes (PA3946-3948, also

termed rocARS) were shown to regulate biosynthesis of Cup

fimbria (93), and mutations in any of these genes resulted in

biofilms with an altered mature structure (94).

Dissemination of Cells fromMulticellular Structures

In addition to the mechanisms involved in biofilm for-

mation, bacteria also possess mechanisms to reduce their

adhesiveness and to break down or modulate the biofilm ma-

trix. Emigration of cells from biofilm communities is neces-

sary to spawn novel communities at new locations, and it may

be induced if the biofilm cells face unfavorable conditions

(e.g., 95,96). Migration of cells may also allow sessile commu-

nities to change spatial organization in response to changing

environments (e.g., 34). After prolonged biofilm development

of P. aeruginosa in flow-chambers local dispersion was

REVIEW ARTICLE

Cytometry Part A � 75A: 90�103, 2009 97

observed as a hollowing out of the mature microcolonies (97).

Through careful microscopic inspection it was observed that

two subpopulations existed in the mature multicellular struc-

tures. The outer parts of these structures contained a wall-

forming subpopulation of non-motile cells, whereas a motile

rapidly moving cell subpopulation was present inside the mul-

ticellular structures. The motile subpopulation coordinately

evacuated the multicellular structures from local break out

points resulting in structures with a central void. This phe-

nomenon has been termed ‘‘seeding dispersal,’’ and was shown

to be dependent on the mature multicellular structures reach-

ing a critical size, suggesting that it may be induced by sub-

strate limitation or accumulation of signal molecules or waste

products. Biosurfactants appears to have multiple roles in

P. aeruginosa biofilm development, as production of large

amounts of rhamnolipid biosurfactant has been associated

with dispersal of cells from P. aeruginosa biofilms (98).

ANTIMICROBIAL ACTION ON BIOFILM CELLS

Biofilms in the environment as well as those associated

with animals and plants or present in man-made environ-

ments are frequently exposed to antimicrobial compounds,

both of natural and synthetic origin. Flow-chamber-grown

biofilms appear to be a useful model system to study antimi-

crobial action in biofilms, for example, as the spatial appear-

ance and distribution of dead and surviving cells in a biofilm

upon antimicrobial attack can be followed in real time. Here

we will give an overview of the spatial antimicrobial suscepti-

bility and tolerance phenotypes of P. aeruginosa biofilms living

in flow-chambers exposed to a number of different anti-

microbial compounds.

As described in the previous section, P. aeruginosa mush-

room-shaped biofilms are commonly found to be composed

of two major subpopulations, a subpopulation situated close

to the substratum and a subpopulation on top. Intriguingly, it

appears that antimicrobial compounds seem to exert their

antimicrobial effects on only one of the two subpopulations,

whereas the other subpopulation survives the treatment. In

most cases the surviving subpopulation of cells exhibits phe-

notypic tolerance and not resistance, as surviving biofilm cells

harvested from antimicrobial-treated biofilms exhibit the

same antimicrobial susceptibility phenotype as the cells, which

were used to initiate the biofilm (e.g., Refs. 41,42).

Effect of Conventional Antibiotics on Biofilm Cells

Most conventional antimicrobial agents used to treat bac-

terial infections in humans and animals, interfere with funda-

mental physiological processes of bacterial cells, such as repli-

cation, transcription or translation processes. Here we describe

the effect of three such conventional antibiotics on mush-

room-like shaped P. aeruginosa biofilms, namely ciprofloxacin,

tetracycline, and tobramycin.

The fluoroquinolone ciprofloxacin induces bacterial cell

death by interfering with the replication process due to inhibi-

tion of the DNA gyrase. Ciprofloxacin is administered for

treatment of various infections caused by Gram-negative and

Gram-positive bacteria. Exposure of P. aeruginosa flow-cell-

grown biofilms was found to preferentially induce cell death

in the cap-forming cell subpopulation (Fig. 5B) (41). Using in

situ gene expression analysis, involving a growth activity-de-

pendent fluorescent reporter (see details about the fluorescent

reporter above), it was found that ciprofloxacin specifically

targets the P. aeruginosa biofilm cells exhibiting high metabolic

activity in the upper part of the multicellular structures. By

contrast, the biofilm cells in the deeper layers exhibiting low

metabolic activity survive ciprofloxacin treatment (Fig. 5B)

(41). A similar phenotype with respect to the distribution of

growth activity and ciprofloxacin-induced cell death was

observed for a P. aeruginosa colony biofilm (99).

Tetracycline is an antimicrobial agent, which originates

from secondary metabolites produced by Streptomyces spp. It

can inhibit bacterial protein synthesis by preventing attach-

ment of aminoacyl-tRNA to the ribosomal acceptor site (A-

site) and thereby induce cell death. When mature P. aeruginosa

wild type biofilms were exposed to tetracycline, the cells of the

cap-forming subpopulation were killed, whereas the cells

situated in the deeper areas survived the treatment (41). An

experiment involving treatment of a biofilm formed by a

strain, which harbors the growth activity-dependent fluores-

cent reporter fusion, indicated that tetracycline preferentially

kills the cells in the upper area, which exhibit high metabolic

activity. By contrast, cells in the deeper areas of the biofilm,

which exhibit a lower metabolic activity, were not killed by

tetracycline (S.J.P. and T.T.N., unpublished observation).

The aminoglycoside tobramycin is a secondary metabo-

lite derived from Streptomyces spp. and it can inhibit protein

synthesis in Gram-negative bacteria by preventing transloca-

tion of peptidyl-tRNA from the A-site to the P-site of the ribo-

some, and thereby induce cell death. When mature P. aerugi-

nosa biofilms were exposed to tobramycin, the cells situated in

the upper area of the multicellular structures were killed,

whereas the cells situated in the deeper areas survived the

treatment (36,100). The efficiency of tobramycin-induced kill-

ing in P. aeruginosa biofilms was increased by co-administra-

tion of furanone C-30, a compound, which was identified to

inhibit quorum sensing regulated gene expression (100). A

biofilm formed by a mutant strain, which was defective in

las- and rhl-mediated cell-to-cell-communication showed

increased sensitivity to tobramycin, indicating a possible role

of quorum sensing in tolerance towards tobramycin (36). Mah

et al. presented results, which indicate that in biofilm cells of

strain P. aeruginosa PA14 periplasmatic glucans might seques-

ter tobramycin and hence prevent the interaction of tobramy-

cin with its target (101). An ndvB-mutant, which is deficient

in the synthesis of periplasmatic glucans, exhibited increased

sensitivity to tobramycin in biofilms (101).

Altogether this indicates, that conventional antimicrobial

compounds, which interfere with fundamental physiological

processes of bacterial cells preferentially induce cell death in

P. aeruginosa biofilm cells that have a high metabolic/physio-

logical activity in the top layer of the multicellular structures.

In contrast, cells that have a low metabolic/physiological

activity are able to survive exposure to the antimicrobial

compound.

REVIEW ARTICLE

98 Insight Into Biofilms via Confocal Microscopy

Effect of Membrane-Targeting Compounds

on Biofilm Cells

The rise in appearance of multiresistant bacteria and the

persistence of biofilms in medical settings has increased the in-

terest in alternative antimicrobial compounds. Among the

new potential antimicrobial therapeutics are antimicrobial

peptides, as resistance to antimicrobial peptides has rarely

been observed so far (102–104). In addition, antimicrobial

peptides are promising new drugs due to their ability to mod-

ulate immune responses (103,105). Antimicrobial peptides are

found to exert their primary antibacterial activities by interfer-

ing with the bacterial membrane, resulting in leakage and

eventual death of the bacterial cell (e.g., 106,107). Here we

describe the effect of the antimicrobial peptide colistin (poly-

myxin E) on P. aeruginosa biofilms. In addition we describe

the effects of two other membrane-targeting compounds on P.

aeruginosa biofilms, namely the detergent sodium dodecyl sul-

fate (SDS) and the chelator ethylenediaminetetraacetic acid

(EDTA).

Colistin belongs to the polymyxin group of antimicrobial

peptides, and it is synthesized by strains of Paenibacillus spp.,

such as P. polymyxa (107–111). Colistin is administered as

treatment against infections caused by Gram-negative bacteria,

for example, pulmonary infections caused by P. aeruginosa in

CF patients, or sepsis, wound infections, and urinary tract

infections caused by a variety of Gram-negative bacteria (112–

115). When mature mushroom-like shaped P. aeruginosa bio-

films were exposed to a clinically relevant concentration of

colistin, only the stalk-forming cell subpopulation was killed,

whereas the cap-forming subpopulation survived the treat-

ment (Fig. 5C) (41,42). Detailed investigations on the spatio-

temporal-dependent effects of colistin exposure on P. aerugi-

nosa biofilms using fluorescent reporters and knock-out

mutants provided evidence, that the metabolic/physiological

active cells of the cap-subpopulation were able to adapt to

colistin by inducing two antimicrobial tolerance mechanisms:

the polymyxin resistance (pmr) LPS-modification system, and

the antimicrobial efflux pump MexAB-OprM (41). In con-

trast, cells with low metabolic/physiological activity in the dee-

per layer of the multicellular structures were not able to adapt

to colistin exposure, and were therefore killed by the action of

the antimicrobial peptide (41). Although tolerance develop-

ment in initial multicellular structures (2-day-grown bio-

films) in addition appeared to be dependent on type IV pili

driven motility (42), cellular migration evidently does not

play a role in colistin-tolerance development in mature multi-

cellular structures (4-day-grown biofilms) (41). Interestingly,

the observed spatial distribution of live and dead cells upon

colistin exposure appears to be independent of the actual

three-dimensional structure of the biofilm and the carbon

source used for biofilm-growth. Mushroom-shaped as well as

irregular-shaped glucose-grown mutant biofilms, and flat-

structured citrate-grown biofilms exhibited a subpopulation

of dead cells close to the substratum and a subpopulation of

live cells in the upper cell layer of the multicellular structures

upon colistin treatment (41,42, and J. Haagensen, personal

communication).

SDS is a synthetic anionic surfactant, and this compound

and its derivatives can be found in many household products

(e.g., soaps). Due to its amphiphilic characteristics SDS inter-

feres with biological membranes and is also known for its abil-

ity to denature proteins. When initial and mature multicellu-

lar structures formed by P. aeruginosa were exposed to SDS,

cell death was induced in the cell subpopulation close to the

substratum, whereas the cells in the top layer of the biofilm

survived the treatment (42). Another study reported disrup-

tion of P. aeruginosa biofilms via hollowing after exposure to

SDS (98). However, as the reported experiment was performed

in the absence of a fluorescent indicator for dead cells (e.g.,

propidium iodide) it is unclear whether cells from the interior

area of the biofilm detached or were killed. The genetic deter-

minants and mechanisms, which facilitate tolerance develop-

ment of the surviving fraction of cells are unknown at present,

and are subject to ongoing research in our laboratory.

EDTA has the ability to form complexes with metal ions,

such as Mg21, Ca21 and Fe31. Exposure of proteobacterial

Figure 4. Determination of metabolic/physiological activity during development of multicellular structures using a growth rate dependent

fluorescent reporter. P. aeruginosa wild type cells harboring a single chromosomal copy of Tn7-rrnBP1-gfp[AGA], expressing a Gfp versionwith a short half life under the control of a ribosomal promoter (A), and, as control, P. aeruginosa wild type cells harboring a single chro-mosomal copy of Tn7-rrnBP1-gfp, expressing a stable version of Gfp under the control of a ribosomal promoter (B), were grown as biofilmand confocal laser scanning micrographs acquired every 24 h for 4 days. The confocal laser scanning micrographs represent sections of

multicellular structures in the yz plane. Reproduced from Mol Microbiol 68:223--240 with permission from John Wiley & Sons, Inc. [Color

figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

REVIEW ARTICLE

Cytometry Part A � 75A: 90�103, 2009 99

cells to EDTA has been reported to result in removal of diva-

lent cations (Mg21, Ca21) from LPS of the outer membrane

and consequently in disruption of the outer membrane

(116,117). When mature multicellular structures formed by P.

aeruginosa were exposed to EDTA, cell death was induced in

the cell subpopulation close to the substratum, whereas the

cells in the top layer of the biofilm survived the treatment

(118). In addition, EDTA exposure was also reported to

induce dispersal of some cells from the multicellular structures

(118). Induction of cell death and dispersal by EDTA was

found to be inhibited by the addition of Mg21, Ca21, or Fe31

ions, supporting the notion, that EDTA exerts its effects on

biofilm cells via complex-formation with metal ions, present

in the outer membrane of the cells and possibly also part of

the stabilizing extracellular matrix (118). The reason for why

the upper subpopulation of cells survived the EDTA treatment

is unclear at present.

Altogether this indicates, that compounds, which inter-

fere with membrane function of bacterial cells preferentially

induce cell death in P. aeruginosa biofilm cells in the deeper

layer of the multicellular structures having a low metabolic/

physiological activity. In contrast, cells in the top layer of the

multicellular structures are able to survive exposure to the

antimicrobial compound.

Effect of Combined Antimicrobial Treatment

on Biofilm Cells

As described above, exposure of P. aeruginosa biofilms to

a single antimicrobial agent was able to kill only a subpopula-

tion of the biofilm cells, whereas the remaining biofilm cells

were able to withstand the action by the particular antimicro-

bial agent. It was observed that conventional antimicrobial

agents preferentially targeted the subpopulation of cells form-

ing the upper layer of the biofilm, whereas membrane target-

ing compounds preferentially targeted the subpopulation of

cells situated in the deeper layer of the multicellular structure

(41). It was therefore of interest to investigate, if a combined

antimicrobial treatment strategy, involving a compound tar-

geting the biofilm cells in the upper layer and a compound tar-

geting the biofilm cells in the deeper layer, could be potentially

successfully used to kill all biofilm cells. Indeed, combined

antimicrobial treatments involving either ciprofloxacin 1colistin, or tetracycline 1 colistin, were found to kill nearly all

biofilm cells. By assessing the number of surviving cells it was

found that in particular the exposure of the P. aeruginosa bio-

film cells to ciprofloxacin 1 colistin was very efficient leaving

only a few single surviving cells (41). Interestingly, it was

observed that combined antimicrobial treatment using cipro-

floxacin 1 colistin in cystic fibrosis (CF) patients is effective

Figure 5. Antimicrobial action on bio-

film cells using single and combined

antimicrobial treatment. Mature multi-

cellular structures by P. aeruginosawild type cells were established within

4 days, and then continuously exposed

for 24 h to either, only propidium

iodide (0.3 lM) as control (A), 60 lg/mlciprofloxacin and propidium iodide (0.3

lM) (B), 25 lg/ml colistin and propi-

dium iodide (0.3 lM) (C), or 60 lg/mlciprofloxacin, 25 lg/ml colistin, andpropidium iodide (0.3 lM) (D). The con-focal laser scanning micrographs show

a section of a multicellular structure in

the yz plane, respectively.

REVIEW ARTICLE

100 Insight Into Biofilms via Confocal Microscopy

in reducing the onset of chronic P. aeruginosa infection (119),

and hence this particular treatment strategy was recently

implemented in the recommended early intervention and pre-

vention therapy in CF (120,121).

CONCLUSION AND FUTURE PROSPECTS

The biofilm mode of living is assumed to be the predomi-

nant bacterial life style in nature. These complex microbial

communities carry out processes in the natural environment as

well as man-made environments, and thereby contribute to

human development, health and disease. Investigations of bio-

films established under controlled conditions in laboratory set-

ups have provided fascinating insights into the fundamental

capacities of bacteria to form multicellular structures. Studies of

model systems, such as flow-chamber-grown biofilms, have

revealed a set of inherent elements of the participating microbes

that can facilitate their organization into multicellular commu-

nities. Among these factors are for example the production of

matrix compounds, cell-surface bound proteins, the secretion

of biosurfactants, cellular migration, and regulatory elements

such as signal transduction systems, and intra- and extra-cellu-

lar signal messenger molecules. CLSM monitoring of fluores-

cently color-coded bacteria, grown in mixed-strain biofilms

under continuous hydrodynamic conditions, has provided in-

triguing insights into the spatiotemporal developmental pro-

cesses, in some cases down to the single cell level (e.g., 30,31).

Detailed microscopic examinations have revealed that, even in

monospecies biofilms, a number of physiologically distinct cell

subpopulations exist and differentiate during biofilm develop-

ment (e.g., 41). Intriguingly, recent investigations have revealed

that these distinct cell subpopulations exhibit differential sensi-

tivity towards antimicrobial agents (e.g., 41,42). One cell subpo-

pulation, which exhibited high metabolic/physiological activity,

was sensitive towards conventional antimicrobial agents,

whereas the second subpopulation was refractory (41). How-

ever, the second cell subpopulation exhibited increased sensitiv-

ity towards a number of membrane-targeting compounds, and

hence a combined antimicrobial treatment using a conventional

antibiotic and a membrane-targeting compound was able to kill

both cell subpopulations of the biofilm (41). These, and other

studies highlight the importance of studying the characteristics

of biofilm cells at the subpopulation, and single cell level.

Future studies, facilitated by sophisticated approaches

and new technologies, will increase our understanding of mi-

crobial life in multicellular communities. Investigations of spa-

tiotemporal gene expression in biofilms by the use of fluores-

cent reporter genes and CLSM will continue, and expand

through the use of combinations of gene expression markers.

Cell subpopulations will be isolated from complex commu-

nities, for example, by the use of microdissection, microfluidic

devices or cell sorting, and subjected to further analysis, such

as RT-PCR measurements of specific gene expression or DNA

array analysis of global gene expression (e.g., Ref. 122). More-

over, new technologies are emerging that might enable analysis

of global transcription profiles of single biofilm cells

(123,124). Studies of the metabolic functions of subpopula-

tions of biofilm cells or single cells in a biofilm community,

might be done by the use of NanoSIMS (nanometer-scale sec-

ondary-ion mass spectrometry) or related techniques, which

have recently been successfully employed on microbial cells

from the environment and microbial cells associated with ani-

mals (e.g., 125,126). Insight into the features of distinct cell

subpopulations and single cells in microbial communities will

provide a more complete understanding of the microbial mul-

ticellular lifestyle, and open up new strategies to manipulate

harmful biofilms to restore and maintain human well-being.

ACKNOWLEDGMENTS

The authors acknowledge the scientific contributions of

their colleagues at the Technical University of Denmark, and

elsewhere.

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