Diapositivo 1

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First International Practical Course in Biofilm
Science 11th -16th September 2006 Braga,
Portugal
University of Minho Department of Biological
Engineering
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P1 - Microscopic analysis of biofilms
Scanning Probe Microscopy Techniques
Light microscopy
AFM
STM
Optical sectioning microscopy
EDIC
Electron Microscopy Techniques
Confocal laser scanning microscopy
(Epi)Fluorescence microscopy
SEM
TEM
Bibliography Nature Methods 2, (01 Dec 2005)
Review
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P1 - Microscopic analysis of biofilms
Visualization of three different procedures under
an EF microscope
PNA FISH
LIVE/DEAD
DAPI
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LIVE/DEAD on S. sciuri
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DAPI on H. pylori
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PNA FISH on H. pylori
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(No Transcript)
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P2 - Assessment of biofilm growth and activity
Crystal Violet Staining
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P2 - Assessment of biofilm growth and activity
Dry Weight
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P2 - Assessment of biofilm growth and activity
In which material the biofilm formation was
higher?
How can you correlate the amount of biofilm
biomass with its activity?
How can you compare the different assays?
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P2 - Assessment of biofilm growth and activity
Try different assays and different conditions and
then choose the one you think is the best and use
it in all your sampples!
What assay should I choose?
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P3 - Biofilm composition

• Biofilm scraping
• Extracellular polymeric substances (EPS)
  extraction
• EPS quantification
• Extracellular proteins analysis.

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P3 - Biofilm composition
98 1 Viable cells
Table 1. Characteristics of the biofilms formed
on the surface of the stainless steel after 6 d
of growth. Mean SD
Parameters analyzed
Figure 1. Epifluorescence photomicrograph of
biofilms formed on stainless steel 6 d of growth.
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P3 - Biofilm composition
FIGURE 2. SEM photomicrographs of a 6 d old
Pseudomonas fluorescens biofilms formed on
stainless steel slides
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P3 - Biofilm composition
Not conclusive results. Chromatography MS/MS
Figure 3. Extracellular proteins entrapped within
a SDS-Page gel. Numbers on the left represent
molecular weights in kDa.
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P3 - Biofilm composition
Different
Similar
Table 2. Characteristics of the biofilms formed
on the surface of the stainless steel cylinders
after 7 d of growth. Mean SD.
Table 1. Characteristics of the biofilms formed
on the surface of the stainless steel after 6 d
of growth. Mean SD
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P4 - Physico-chemical properties of surfaces and
cells
P4.1 Determination of surface tension components
and hydrophobicity through Contact angles
technique
Table 1 - Contact angle (in degrees), surface
tension components (?LW, ?, ?-, ?AB, ?TOT) and
degree of hydrophobicity (?G1w1) of bacterial
cells (in mJ/m2).
?W - Contact angle of water ?F - Contact angle
of formamide ??-B - Contact angle of
a-bromonaphtalene
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P4 - Physico-chemical properties of surfaces and
cells
P4.1 Determination of surface tension components
and hydrophobicity through Contact angles
technique

• Table 2 - Contact angle (in degrees), surface
  tension components (?LW, ?, ?-, ?AB, ?TOT) and
  degree of hydrophobicity (?G1w1) of the materials
  assayed (in mJ/m2).

?W - Contact angle of water ?F - Contact angle
of formamide ??-B - Contact angle of
a-bromonaphtalene
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P4 - Physico-chemical properties of surfaces and
cells
P4.2 Determination of hydrophobicity through MATH
technique
Table 3- Percentage of adhered cells to the
hydrocarbon as measured by the MATH technique.