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Biofilm Analysis

Hans-Curt Flemming
Jost Wingender
Martin Strathmann

Institute of Interface Biotechnology

University of Duisburg-Essen
Many Questions in Biofilm Analysis
Ö Where are the biofilms?
Ö How can they be sampled?
Ö How many cells are there?
- Biomass
- Numbers (cfu, cells per g, per mL or per cm²)
- Viability, activity
ÖBiodiversity: Which organisms are there?
Ö Biofilm structure: How are they organized
Ö Activity: What do they do?
- Growth rate
- Spectrum of utilized substrates
- Products
- Effect on environment
Biofilm sampling:
On surfaces!

! Scratching, scratching, scratching (razor blades,

rubber scrapers, cotton swabs etc.)
! Removal of parts of support material with
biofilms ⇒ laboratory
! Expose test surfaces („coupons“), remove after
given time ⇒ laboratory
Sample intact or homogenized
" Maximum number: nucleic acid stains (acridin
orange, DAPI, but: no differentiation live/dead
" Active organisms:
- cultivation methods on various media
- determination of enzymatic activity, incl. redox
- determination of elongation
- turbidity
" Indirect methods:
- oxygen consumption
- increase in conductivity
- CO2 production
- production of other metabolites (nitrite, sulfate)
The problem with
colony forming units
(cfu): many cells do
not grow with the
substrates offered

SEM of a colony

SEM of agar of
space between
The problem with quantification or:
Which cell number do you want?

Quantitative values: different methods, same sample

Microscopical biofilm analysis
h Light microscopy, epifluorescence
h Confocal Laser Scanning microscopy (CLSM)
h Scanning electron microscopy (SEM)
h Environmental SEM
h Transmission electron microscopy (TEM)
h Atomic force microscopy (AFM)
Biofilm on rubber valve in a drinking water system

Light microscopy, phase

(1000 x)

Scanning electron micrograph

of same sample
(courtesy of G. Schaule)
Microscopic Details of SSF - Bacteria
Microscopic view of the Schmutzdecke,
stained with DAPI, 1000x magnification,
epifluorescence microscopy



Bacteria as substrate of nutrient chain in the schmutzdecke

Flocs from Elbe river (DAPI)


Rods (1000x) Spirillae (1000x)

microscopy on sand:
High auto-fluorescence
of support material

Biofilm on
quartz sand
Mature biofilms in biofouling
cases, displaying varying
biofilm morphologies:
Reverse osmosis membranes,
irreversibly blocked by biofilms
which have survived many
cleaning and „disinfection“
The confocal laser scanning microscope (CLSM) -

Photomultiplier (PMT)



Beam Splitter
Objective Lens

Confocal Laser Scanning
Microscopy (CLSM)
Non-destructive, 3-dimensional
information about biofilm architecture

Vertical section
a b

a b
Confocal laser scanning microscopy (CLSM)
3-D micrographs of a P. aeruginosa biofilm with lectin Concanavalin A (red,
alginate spec.) and SYTO 9 (green, cells), magnification 1000 x

a) P. aeruginosa SG81 (mucoid): thick (40 µm), heterogeneous biofilm, lots of

alginate between cells
b) P. aeruginosa SG81R1 (non mucoid): thin (20 µm), unstructured biofilm, cells
densely packed
AFM picture of E. coli .../research/research.htm
Vitality staining: CTC BRespiration activity

Left: DAPI Right: CTC, same sample

CTC: 5-cyano-2,3-ditolyl tetrazolium chloride
CTC is a tetrazolium salt which can be used as an e-- acceptor
The oxidized form is water soluble and colourless
The reduced form precipitates as fluorescent crystals
Flow cell:
A suitable tool for analysis of biofilm
processes directly observed by microscopy
Analysis of Biofilm Structure: Influence of alginate,
investigated by Confocal Laser Scanning Microscopy

aeruginosa SG 81
mucoid strain, grown
in a flow-through cell

aeruginosa SG 81 R
Non-mucoid mutant
Original CLSM picture: „hanging biofilm“

Biofilm of P.
aeruginosa after 5
days („Mushroom
(Center for Biofilm
Engineering, Bozeman)
Microelectrode measurement:
Oxygen gradient profile in a biofilm
(de Beer et al., 1994)
Principles of ATR-FTIR spectroscopy
• ATR = Attenuated Total Reflectance
• The evanescent wave penetrates only a small distance through the
crystal (typically about a micrometre), depending on the refractive index
of the IR-transparent crystal, the internal reflectance element (IRE).
• By constructing flow chambers on either side of the IRE, a biofilm can
be grown on the crystal, allowing measurement of the IR spectrum of
the biofilm. Water must be subtracted from the spectrum.

sampled region
Non-destructive investigation of biofilm
formation during first few days by FTIR-
ATR spectroscop (Schmitt and Flemming, 1998)
Question in Biofilm Population Analysis:
who is who?

Light microscopy of environmental sample (T. Staley)

Classical population analysis:
hIsolation of organisms from population
hEnrichment and cultivation on various media
hDifferentiation by growth media, reactions,
enzymes, utilization of substrates etc.

•Selective isolation
•Only cultivable organisms will grow
•Most organisms do not grow on those media
The main molecular Mixed
techniques used in microbial
Fluorescent in-situ
biofilm population (FISH)
Step 1 Extract DNA

Total community DNA

Step 2 PCR amplify a representative gene,

Cultivation usually the 16S rRNA gene
Mixture of total community 16S rRNA genes
Step 3 Separate and organise the
16S rRNA genes
Find 85 - 95%
of bacteria present samples
in sample Create a
clone library

Step 4 Sequence clones Sequence individual bands

FISH for sand filter biofilm analysis
• in situ identification and quantification of bacteria groups
by fluorescence-labelled probes
• However: you can only detect what you have probes for
DAPI (background staining) FISH

Supernatant of sediment sample stained with DAPI and a Cy3 labelled

EUB338 probe
Red arrows: cells which are NOT eubacteria
Application of FISH directly in biofilms

Biofilm in phase contrast

Same sample: E. coli in biofilm, Grazing pressure on bacteria by

visualized by FISH amoebae, visualized by FISH probes
Yellow: Amoebae
Green: Bacteria

(Courtesy of U. Szewzyk, TU Berlin)

Investigation of river snow (microbial aggregates) using FISH
Böckelmann et al 2002

G = Autofluorescent algae are blue and

lectin is stained green
H = false coloured algae are red, lectin is
green and bacteria stained with FISH probe
EUB338 are red. See small clusters of
bacteria embedded in lectin.
I = FISH probe EUB338 are red, lectin is
green. Some bacteria are coated in lectin
and some not.
J = 2 small microcolonies of bacteria
stained with FISH probe EUB338 (red) and
surrounded by green lectin.
K = FISH probe EUB338 (red) stained pure
culture with green stained lectin.
L = close up of K indicating that FISH gives
a clear hybridisation signal with the bacteria
and that the lectin staining enables it to be
visualised and its location in relation to the
cells to be determined in situ
Legionella in amoebae

U. Szewzyk
Problems with gene probes in
environmental biofilms
h Background fluorescence of substratum
h Fluorescent particles
h Dormant organisms: low activity and low
h Uneven penetration of probes through the
bacterial cell wall
h Fixing and dehydration of samples may change
the spatial arrangement of microconsortia
h Steric hindering of specific binding of nucleotide
sequence to target
EPS, a biofilm component
h Physical isolation methods
- Centrifugation (high and low speed)
- Ultrasonication and centrifugation
- Stirring (Ultrathurrax, 20.000 rpm, 60 sec)
- Boiling (30 min) or steam (autoclave 10 min)

h Physical and chemical isolation methods

- Ion exchanger (Dowex) and stirring
- NaCl (8,5 %), Formaldehyde (0,22 %), ultrasonication
- Phenol/water extraction with intermittent, ultrasonication

h Chemical isolation methods

- Ethanol extraction
- NaOH/EDTA treatment
- Dowex ion exchanger
- Crown ether complexation of Ca2+
- Phenol/Water extraction (lipopolysaccharides)

@ Important: Integrity of cells. Test for cytosol leaking:

Activity of G-6-P-Dehydrogenase, a strictly intracellular enzyme
Cultivation of biofilms on agar surface EPS: a biofilm
or membrane filters (PIA, 36 °C, 24 h–72 h)
Isolation from a mucoid
suspension of biofilms in 0.14 M NaCl biofilm of Pseudomonas

(40 000 g, 10 °C, 2 h)

membrane filtration of supernatant Test for integrity of cells: activity of

(0.2 µm pore diameter) G-6-P-dehydrogenase

Dialysis → freeze-drying → EPS

cell-free EPS solution precipitation → enzyme treatment → Alginate
Separate visualisation of EPS and cells
red: alginate-bound fluorescent ConA
green: P. aeruginosa cells, Syto 9
Selective visualization of proteins by
SYPRO Ruby dye, a fluorophor

a b

Binding of SYPRO Ruby to 24 h old biofilms of P. aeruginosa SG81

(a) 1000 x magnification,
(b) 4000 x magnification.
CLSM micrographs
Visualization of enzyme activity by enzyme-linked
fluorophors (ELF) substrates
24 h 48 h 72 h

Example 1: Extracellular lipase activity

Red: cells
Green: ELF substrate precipitated
Visualization of enzyme activity in biofilms

a b c

Example 2: Extracellular redox enzymes

Visualized by CTC reduction (red)
Cells: green (SYTO 9)
Bar: 10 µm, magnification 1000 x

Æ Redox-Enzymes demonstrated in the EPS matrix in distinct clusters

Æ Homogenous distribution in the matrix
Æ Spatial and temporal heterogeneity
Some specific chromophores for visualization of
biofilm biopolymers
• Polysaccharides Tagged lectin (ConA) Æ alginate
Calcofluor White Æ alginate

• Proteins (total) SYPRO Ruby protein gel stain

h Enzymes ELF®-97-Substrates
Æ Phosphatases, Esterases, Lipases
CTC Æ Redox-Enzymes

• Lipids DiD Æ Zellmembranes + Vesicles

• Hydrophobic dyes Nile red Æ cell membranes and intracellular

Oil red Æ cell membranes
Biofilm Analysis
biofilm sample

Analytical components & structure & processes &

question properties architecture interactions

- Chemical analyses - Confocal microscopy -Flow cells with CLSM

- Biochemical analyses - SEM * time lapse microscopy
* Proteomics - ESEM * reporter genes
* Enzymes - TEM * GFP tagged organisms
- Population analysis - AFM - Microelectrodes
* classic microbiology - Specific staining * Oxygen
Methods * Fatty acid analysis of components * Nitrate, ammonia etc.
* Molecular biology • Cells * CO2
- Physicochemical • EPS * H2S
properties • Enzymes - Autoradiography & FISH
* Mechanical stability • Sorbed substances
* Diffusivity • Vesicles
* Sorption properties
Combined Strategy: Microscopy and Biochemistry
Acquired information:
3D-distribution of dyes in biofilm matrix
In-situ analysis
Æ Architecture

In-situ enzyme
Relevance of possible target components
treatment and for observed binding reaction

In vitro-Analysis of Interaction of dye with target molecules

dye binding at Æ Quantification and specifity
target components

Interaction of dyes with biopolymers
investigation of Æ Specifity
dye binding

Quantitative Quantitative composition of biofilm

biochem. analysis and EPS
Which method yields what?