Académique Documents
Professionnel Documents
Culture Documents
Hans-Curt Flemming
Jost Wingender
Martin Strathmann
SEM of a colony
SEM of agar of
space between
colonies
The problem with quantification or:
Which cell number do you want?
rods
spirillae
(1000x)
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“
cycles
The confocal laser scanning microscope (CLSM) -
overview
Photomultiplier (PMT)
Pinhole
Laser
Beam Splitter
Scanner
Objective Lens
Z-control
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
chemiris.chem.binghamton.edu:8080/ .../research/research.htm
Vitality staining: CTC BRespiration activity
Pseudomonas
aeruginosa SG 81
mucoid strain, grown
in a flow-through cell
Pseudomonas
aeruginosa SG 81 R
Non-mucoid mutant
Original CLSM picture: „hanging biofilm“
Biofilm of P.
aeruginosa after 5
days („Mushroom
model“)
(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?
Problems:
•Selective isolation
•Only cultivable organisms will grow
•Most organisms do not grow on those media
The main molecular Mixed
techniques used in microbial
community
Fluorescent in-situ
Hybridisation
biofilm population (FISH)
analysis
Step 1 Extract DNA
U. Szewzyk
Problems with gene probes in
environmental biofilms
h Background fluorescence of substratum
h Fluorescent particles
h Dormant organisms: low activity and low
hybridization
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)
centrifugation
(40 000 g, 10 °C, 2 h)
a b
a b c
In-situ enzyme
Relevance of possible target components
treatment and for observed binding reaction
Mikroscopy
Spectroscopical
Interaction of dyes with biopolymers
investigation of Æ Specifity
dye binding